Cancer specific promoters

ABSTRACT

The present invention regards cancer-specific control sequences that direct expression of a polynucleotide encoding a therapeutic gene product for treatment of the cancer. Specifically, the invention encompasses breast cancer-, prostate cancer-, and pancreatic cancer-specific control sequences. Two breast cancer-specific sequences utilize specific regions of topoisomerase IIα and transferrin receptor promoters, particularly in combination with an enhancer. The prostate cancer-specific and pancreatic cancer-specific control sequences utilize composites of tissue-specific control sequences, a two-step transcription amplification sequence, and a post-transcriptional control sequence. In more particular embodiments, these polynucleotides are administered in combination with liposomes.

The present invention claims priority to U.S. Provisional ApplicationSer. No. 60/559,111, filed Apr. 2, 2004, which is incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention is directed to the fields of cell biology,molecular biology, cancer biology, and medicine. More particularly, thepresent invention regards cancer-specific regulatory sequences forregulation of expression of a therapeutic polynucleotide useful forcancer therapy.

BACKGROUND OF THE INVENTION

The ability to control expression of particular polynucleotides upongene transfer is a useful function, particularly for applications wherespecific localized activity is desired. Such is the case for cancer,where it is prudent to confine destructive or lethal gene products tothe cancerous cells while preventing at least in part such activity innormal cells.

Breast Cancer Tissue-Specific Expression

Current breast cancer therapies, such as chemotherapy (CT) andradiotherapy, have low selectivity for tumor cells and side effects fornormal tissues. To minimize the side effects, these therapies aregenerally given in an intermittent manner, allowing normal cells torecover between treatment cycles. However, during the recovery period,some surviving cancer cells become more resistant to the treatmentbecause of gene mutation. Consequently, cancer recurrence or progressionmay occur. Tumor-targeting gene therapy minimizes treatment side effectsand the risk of developing resistance by acting on the tumor-specificsignaling pathways.

One breast cancer-specific promoter described herein comprises selectedportions of the topoisomerase IIα gene. Although the 5′ flanking regionof the topoisomerase IIα gene has been known for some time Hochhauser etal., 1992), a particular active region described herein has not beendemonstrated to be useful for breast cancer tissue, even when linked tocytomegalovirus enhancer (Mo et al., 1998).

Another breast cancer-specific promoter described herein comprisesselected portions of the transferrin receptor promoter. Transferrinreceptor expression has been localized in breast tissue (Fuernkranz etal., 1991; Shterman et al., 1991) and in breast cancer (Bauman et al.,1997; Shindelman et al., 1981), but a particular region that providessuch activity has not been disclosed.

Prostate Cancer Tissue-Specific Expression

Prostate-specific promoters, such as PSA, probasin, and hK2, have beenrecently developed. The activities of these promoters areandrogen-dependent and the use of androgen-responsive vectors to directexpression of therapeutic genes to prostatic tissue is helpful fornumerous disease stages. Although such prostate-specific promotersresponsive to androgen receptor have been developed by the presentinventors (Xie et al., Cancer Res 2001) and other groups (Zhang et al.,Mol Endocrinol 2000), these androgen-dependent promoters may be lessactive after castration or androgen ablation therapy, which are the mainmodalities for progressive prostate cancer treatment. Patients treatedwith compositions comprising these promoters may fail this kind oftherapy and die of recurrent androgen-independent prostate cancer(AIPC). A novel promoter for prostate cancer gene therapy that will beactive in both ADPC and AIPC to treat metastatic and recurrent hormonalrefractory prostate cancer is lacking in the art.

Pancreatic Cancer Tissue-Specific Expression

Pancreatic cancer is one of the most aggressive human malignancies andthe fifth cause of cancer death, given that no effective modalities areavailable. The present invention addresses such a need by providing apromoter effective to regulate expression of a therapeuticpolynucleotide specifically in pancreatic cancer cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel tissue-specific promoters forregulation of expression of a therapeutic polynucleotide. Thesetherapeutic compositions and methods that utilize them are helpful forcancer treatment, and a skilled artisan recognizes that any additionalmeans in an arsenal to fight cancer is beneficial to public health.

In particular, the invention provides compositions, such astherapeutics, and methods of using same directed to cancer-specificregulated expression of a therapeutic polynucleotide in gene therapy forcancer, such as at least ovarian, breast, pancreatic, and prostatecancer, for example.

Thus, the present invention generally relates to methods for inhibitingproliferation in a cancer cell and/or tumor cell, the method comprisingcontacting the cell with a therapeutic polypeptide in an amounteffective to inhibit proliferation utilizing a cancer-specific promoter,such as one described herein. Inhibition of proliferation may beindicated by, for example, an induction of apoptosis of a cell, such as,for example, in cell culture, inhibition of growth of a cancer cellline, reduction in size of a tumor, and/or an increase in survivability,in exemplary embodiments. More preferably, in some embodiments the cellin which proliferation is to be inhibited is a cell in a livingorganism, for example a human. The inhibition of such transformation hasgreat utility in the prevention and/or treatment of suchtransformation-driven events as cancer, tumorigenesis, and/ormetastasis.

The present invention encompasses polynucleotide constructs comprisingcontrol sequences that direct expression of a therapeutic polynucleotidein a particular tissue and/or type of cell. The polynucleotide may becontacted with or introduced to a cell through any of a variety ofmanners known to those of skill. The therapeutic polynucleotide may beintroduced through direct introduction of the polynucleotide to a cellor tissue of interest. In this case, the therapeutic polynucleotide maybe obtained through any method known in the art.

In specific aspects of the invention, RNA or DNA comprising thetherapeutic polynucleotide may be introduced to the cell by any mannerknown in the art. In certain preferred embodiments, the therapeuticpolynucleotide is introduced into the cell through the introduction of aDNA segment that encodes the therapeutic gene product. In some suchembodiments, it is envisioned that the DNA segment comprising thetherapeutic polynucleotide is operatively linked to the inventivecontrol sequences. The construction of such gene/control sequence DNAconstructs is well-known within the art and is described in detailherein.

In certain embodiments for introduction, the DNA segment may be locatedon a vector, for example, a plasmid vector or a viral vector. The virusvector may be, for example, retrovirus, adenovirus, herpesvirus, vaccinavirus, and adeno-associated virus. Such a DNA segment may be used in avariety of methods related to the invention. The vector may be used todeliver a mutant bik polynucleotide to a cell in one of the gene-therapyembodiments of the invention, in specific embodiments. Also, suchvectors can be used to transform cultured cells, and such cultured cellscould be used, inter alia, for the expression of mutant Bik in vitro.

A skilled artisan recognizes that the promoters of the invention areuseful in any context, including non-cancerous cell-specific expressionor even expression of a polynucleotide that is not cell- ortissue-specific in nature.

In a particular embodiment, a therapeutic gene product is effective onthe respective breast, pancreatic, or prostate cancer tissue. Inexemplary embodiments, the present invention is useful for deliveringgenetic constructs that treat cancers that are estrogen receptorpositive, EGF receptor overexpressing, Her2/neu-overexpressing,Her-2/neu-nonoverexpressing, Akt overexpressing, androgen dependent,and/or angrogen independent, for example. That is, the therapeutic geneproduct is effective on the respective cancer cells regardless of theirstatus of oncogene overexpression, such as Her-2/neu, EGFR, AKT, orregardless of whether their growth is hormone dependent (such as, forexample, MCF-7) or not (such as, for example, PC3).

A skilled artisan is aware of publicly available databases that providetherapeutic polynucleotide sequences, such as the National Center forBiotechnology Information's GenBank database or commercially availabledatabases, such as from Celera Genomics, Inc. (Rockville, Md.). Althoughthere are a plethora of therapeutic polynucleotides that are known inthe art that are later discovered that may be utilized in the invention,some examples include inhibitors of cellular proliferation, regulatorsof programmed cell death, tumor suppressors and antisense sequences ofinducers of cellular proliferation. The therapeutic polynucleotide mayencode small interfering RNAs or antisense sequences, for example.Particular exemplary therapeutic polynucleotides include those thatencode mutant Bik, retinoblastoma, Blk, IL-12, IL-10, IFN-a, cytosinedeaminase, GM-CSF, E1A, p53, and other pro-apoptotic proteins, forexample. Also, a construct may comprise such therapeutic polynucleotidesas TNFα or p53 or inducers of apoptosis including, but not limited to,Bik, p53, Bax, Bak, Bcl-x, Bad, Bim, Bok, Bid, Harakiri, Ad E1B, Bad andICE-CED3 proteases. In specific aspects of the invention, a mutant Bikpolynucleotide encoding an amino acid substitution at threonine 33,serine 35, or both is utilized. In particular aspects of theseembodiments, the amino acids of the mutant Bik polypeptide aresubstituted with aspartate. In other particular aspects, one or morephosphorylation sites are defective in a mutant Bik. In additionalembodiments, the mutant Bik retains anti-cell proliferative and/orpro-apoptotic activity.

In particular embodiments, a construct comprising the inventivetherapeutic polynucleotide and respective cancer-specific controlsequences is introduced into a cell that is a human cell. In manyembodiments the cell is a tumor cell. In some presently preferredembodiments, the tumor cell is a breast tumor cell, an ovarian tumorcell, a prostrate tumor cell, or a pancreatic tumor cell. In someembodiments, a construct comprising the therapeutic polynucleotide andrespective cancer-specific control sequences is introduced by injection.In particular embodiments, the construct comprising the therapeuticpolynucleotide and respective cancer-specific control sequences iscomprised in a liposome.

In some embodiments of the present invention, a construct comprising thetherapeutic polynucleotide and respective cancer-specific controlsequences is used in combination with otheranti-transformation/anti-cancer therapies. These other therapies may beknown at the time of this application, or may become apparent after thedate of this application. A construct comprising the therapeuticpolynucleotide and respective cancer-specific control sequences may beused in combination with other therapeutic polypeptides, polynucleotidesencoding other therapeutic polypeptides, chemotherapeutic agents,surgical methods, or radiation, for example.

A construct comprising the therapeutic polynucleotide and respectivecancer-specific control sequences may be used in conjunction with anysuitable chemotherapeutic agent. In one representative embodiment, thechemotherapeutic agent is Taxol. A construct comprising the therapeuticpolynucleotide and respective cancer-specific control sequences also maybe used in conjunction with radiotherapy. The type of ionizing radiationconstituting the radiotherapy may comprise x-rays, γ-rays, andmicrowaves, for example. In certain embodiments, the ionizing radiationmay be delivered by external beam irradiation or by administration of aradionuclide. The cancer-specific control sequence-regulated therapeuticgene product also may be used with other gene-therapy regimes. Inparticular embodiments, the construct comprising the therapeuticpolynucleotide and respective cancer-specific control sequences isintroduced into a tumor. The tumor may be in an animal, in particular, amammal, such as a human.

Constructs having the inventive tissue-specific promoters regulatingexpression of a therapeutic gene product and polynucleotides of thepresent invention may also be introduced using any suitable method. A“suitable method” of introduction is one that places a therapeutic geneproduct in a position to reduce the proliferation of a tumor cell,preferably in the tissue or cells of interest and/or to ameliorate atleast one cancer symptom. For example, injection, oral, and inhalationmethods may be employed, with the skilled artisan being able todetermine an appropriate method of introduction for a givencircumstance, and the tissue-specific control sequences of the presentinvention direct expression of the therapeutic polynucleotide at leastprimarily in the tissue or cells of interest. In the embodiments whereinjection will be used, this injection may be intravenous,intraperitoneal, intramuscular, subcutaneous, intratumoral, orintrapleural, for example, or of any other appropriate form.

In certain other aspects of the present invention, there are providedtherapeutic kits comprising in a suitable container a pharmaceuticalformulation of a construct comprising the inventive control sequences.In additional aspects, a polynucleotide comprising the inventive controlsequences comprises one or more cloning sites such that a desiredpolynucleotide, such as a polynucleotide of interest, may be cloned intothe site. In particular embodiments, in a polynucleotide having a 5′ to3′ orientation the one or more cloning sites may be located 5′ ofcontrol sequence or 3′ of the control sequence. In additional aspects,one or more therapeutic polynucleotides are also comprised in the kit,such as on the same nucleic acid molecule as the control sequences ofthe present invention. Such a kit may further comprise a pharmaceuticalformulation of a therapeutic polypeptide, polynucleotide encoding atherapeutic polypeptide, and/or chemotherapeutic agent.

The anti-tumor activity, anti-cell proliferation activity, and/orpro-apoptotic activity provided by the gene product of the therapeuticpolynucleotide may be useful for an organism other than the one fromwhich the therapeutic polynucleotide is derived. For example, a murinetherapeutic polynucleotide may be used alternatively or in addition forhuman treatment.

Thus, the present invention provides cancer-specific control sequencesfor targeted expression of a therapeutic polynucleotide, and, therefore,the present invention is directed to a novel improvement to the overallarts of cell growth control, including inhibition of cell proliferationand/or facilitation of cell death. In a specific embodiment, theinhibition of a cell proliferation comprises a delay in its rate ofproliferation, a delay in its total cell numbers of proliferation, orboth.

In an additional object of the present invention, there is a method ofpreventing growth of a cell in an individual comprising the step ofadministering to the individual a construct comprising cancer-specificcontrol sequences that regulate expression of a therapeuticpolynucleotide. In another specific embodiment, the administration ofthe construct comprising the inventive controls sequences is by aliposome.

In another object of the present invention, there is a method ofpreventing growth of a cell in an individual comprising the step ofadministering to the individual a nucleic acid comprising atissue-specific control sequence encompassed by the present invention.In another specific embodiment, the administration of the nucleic acidis by a vector selected from the group consisting of a plasmid, aretroviral vector, an adenoviral vector, an adeno-associated viralvector, a liposome, and a combination thereof. The compositioncomprising the nucleic acid may be dispersed in a pharmacologicallyacceptable excipient, and the composition may be administered to ananimal having a proliferative cell disorder.

In a further object of the invention, a therapeutic polynucleotide isregulated by a tissue-specific promoter, such as one that targetscancerous tissue. Although any promoter that targets cancerous tissuepreferentially over non-cancerous tissue, in a specific embodiment thecancer-specific promoter is a breast cancer specific promoter, aprostate cancer-specific promoter, or a pancreatic-specific promoter,for example.

In a particular embodiment, a breast cancer-specific promoter comprisesa breast cancer-specific sequence and, in further embodiments, anenhancer sequence that augments expression, such as the expressionlevel, of the tissue-specific sequence. In a particular embodiment, aCMV promoter enhancer sequence is linked with a breast cancer-specificsegment from the exemplary topoisomerase IIα (topoIIα) promoter or theexemplary transferrin receptor promoter. The inventors show herein thatboth of these composite promoters drive gene expression selectively inbreast cancer cells and possess activity comparable to the CMV promoter.They are useful for gene targeting to target and treat primary andmetastatic breast cancers with less toxicity to normal tissues.

In another embodiment, the expression of a therapeutic polynucleotide isregulated by a pancreatic-cancer specific promoter. In a particularembodiment, a novel pancreatic cancer specific promoter is utilized,such as one referred to herein as CTP, which is comprised of the minimalCholecystokinin A receptor (CCKAR, −726 to +1 nucleotides) and thepost-transcriptional regulatory element of the woodchuck hepatitis virus(WPRE). This engineered construct has a strong promoter activity anddemonstrates specificity to pancreatic cancer cells in vitro and invivo.

In another specific embodiment of the present invention, a prostatecancer-specific promoter regulates expression of a therapeuticpolynucleotide. In a particular embodiment, the invention utilizes anovel prostate cancer specific promoter, such as one referred to hereinas ATTP, comprised of the minimal human telomerase reverse transcriptasepromoter (hTert), the post transcriptional regulatory element of thewoodchuck hepatitis virus (WPRE), and the ARR2 element derived fromplasmid ARR2PB, which is responsive to androgen stimulation. Thisengineered construct has a strong promoter activity and demonstratesspecificity to both androgen-dependent and androgen-independent prostatecancer cells in vitro. This promoter can be used to specifically drivegene expression of a therapeutic polynucleotide in prostate cancer invivo.

In one object of the invention, there is a polynucleotide constructcomprising a breast cancer-specific control sequence, said controlsequence comprising a selected portion of the topoisomerase IIα promoteror a selected portion of the transferrin receptor promoter. Inparticular, the control sequence comprises a selected portion of thetopoisomerase IIα promoter, such as one comprising SEQ ID NO:12. Thecontrol sequence can also comprise a selected portion of the transferrinreceptor promoter, such as one comprising SEQ ID NO:13.

In particular embodiments, constructs of the present invention comprisean enhancer, such as cytomegalovirus (CMV) enhancer,Glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH), or theβ-actin promoter. The construct may further comprise apost-transcriptional regulatory sequence, such as, for example,woodchuck hepatitis virus post-transcriptional regulatory element(WPRE). In additional embodiments, a construct of the present inventioncomprises a two-step transcriptional amplification (TSTA) sequence,wherein the TSTA sequence includes a DNA binding domain, such as Gal1,Gal4, or LexA, and an activation domain, such as VP2 or VP16, forexample. In particular aspects of the invention, the TSTA sequence isGAL4-VP2 or GAL4-VP16, for example.

In other particular embodiments, the control sequence is operably linkedto a polynucleotide encoding a therapeutic gene product, such as onethat is an inhibitor of cell proliferation, a regulator of programmedcell death, or a tumor suppressor, or one encompassing two of more ofthese activities. Constructs of the present invention may be comprisedin a liposome.

In another object of the invention, a polynucleotide construct comprisesa prostate cancer-specific control sequence that comprises at least twoof the following sequences: a prostate tissue-specific control sequence;a cancer-specific control sequence; and a two-step transcriptionalamplification (TSTA) sequence, said TSTA sequence including a DNAbinding domain and an activation domain. In particular embodiments, theprostate tissue-specific control sequence comprises SEQ ID NO:17. Inother particular embodiments, the cancer-specific control sequencecomprises SEQ ID NO:18. Again, the DNA binding domain of the TSTAsequence may be Gal1, Gal4, or LexA, and the activation domain may beVP2 or VP16. In particular, the TSTA sequence is GAL4-VP2 or GAL4-VP16.

In a specific aspect of the invention, a polynucleotide construct thatcomprises a prostate cancer-specific control sequence further comprisesa post-transcriptional regulatory sequence, such as woodchuck hepatitisvirus post-transcriptional regulatory element (WPRE) sequence. Thecontrol sequence is operably linked to a polynucleotide encoding atherapeutic gene product, in some embodiments, such as an inhibitor ofcell proliferation, a regulator of programmed cell death, or a tumorsuppressor, or one encompassing two or more of these activities. Thepolynucleotide construct comprising a prostate cancer-specific controlsequence may be comprised in a liposome.

In an additional object of the invention, there is a polynucleotideconstruct comprising a pancreatic cancer-specific control sequencecomprising: a pancreatic tissue-specific control sequence, such as, forexample, one comprising SEQ ID NO:14; and a two-step transcriptionalamplification (TSTA) sequence, said TSTA sequence including a DNAbinding domain and an activation domain. In the polynucleotide constructcomprising a pancreatic cancer-specific control sequence, the DNAbinding domain of the TSTA can be Gal1, Gal4, or LexA, and theactivation domain of the TSTA can be VP2 or VP16. In particular, theTSTA sequence is GAL4-VP2 or GAL4-VP 16.

In particular, the construct may comprise a post-transcriptionalregulatory sequence, such as the woodchuck hepatitis viruspost-transcriptional regulatory element (WPRE). The control sequence canbe operably linked to a polynucleotide encoding a therapeutic geneproduct, such as one that is an inhibitor of cell proliferation, aregulator of programmed cell death, or a tumor suppressor, or oneencompassing one or more of these activities. The construct may becomprise in a liposome.

In an additional object of the invention, there is a method ofinhibiting breast cancer cell proliferation, comprising contacting abreast cancer cell with an effective amount of a polynucleotideconstruct that comprises a selected portion of the topoisomerase IIαpromoter or a selected portion of the transferrin receptor, wherein theselected portion may be operably linked to a polynucleotide encoding agene product effective to inhibit the cell proliferation. The selectedportion of the topoisomerase IIα promoter may comprise, for example, SEQID NO:12. The selected portion of the transferrin receptor may compriseSEQ ID NO:13, for example. In particular aspects of the invention, theconstruct further comprises an enhancer, such as CMV,Glyceraldehyde-3-phosphate dehydrogenase promoter (GAPDH), or theβ-actin promoter.

In another object of the invention, there is a method of inhibitingprostate cancer cell proliferation, comprising contacting a prostatecancer cell with an effective amount of a polynucleotide constructhaving at least two of the following sequences: a prostate cell-specificcontrol sequence; a two-step transcriptional amplification sequence; anda cancer cell-specific sequence, wherein the sequences are operablylinked to a polynucleotide encoding a gene product effective to inhibitthe prostate cancer cell proliferation. The construct may furthercomprise a post-transcriptional control sequence operably linked to thepolynucleotide encoding a gene product effective to inhibit the prostatecancer cell proliferation, such as a WPRE sequence, for example.

In an additional object of the invention, there is a method ofinhibiting pancreatic cancer cell proliferation, comprising contacting apancreatic cancer cell with an effective amount of a polynucleotideconstruct comprising a pancreatic cell-specific sequence and a two-stepamplification sequence, both of which are operably linked to apolynucleotide encoding a gene product effective to inhibit the cellproliferation. The construct may further comprise a post-transcriptionalcontrol sequence operably linked to the polynucleotide encoding a geneproduct effective to inhibit the cell proliferation, such as a WPREsequence, for example.

In a further object of the invention, there is a method of treatingbreast cancer in an individual having the cancer, comprising contactingat least one breast cancer cell of the individual with a therapeuticallyeffective amount of a polynucleotide construct comprising a selectedportion of the topoisomerase IIα promoter or a selected portion of thetransferrin receptor, wherein the selected portion is operably linked toa polynucleotide encoding a gene product effective to treat breastcancer. The construct may comprise a selected portion of thetopoisomerase IIα promoter being one that comprises SEQ ID NO:12. Theconstruct may also comprise a selected portion of the transferrinreceptor promoter, such as one comprising SEQ ID NO:13. The constructmay further comprise an enhancer, such as CMV enhancer, and thepolynucleotide may be comprised in a liposome.

In another object of the present invention, there is a method oftreating prostate cancer in an individual having the cancer, comprisingcontacting at least one prostate cancer cell of the individual with atherapeutically effective amount of a polynucleotide constructcomprising at least two of the following: a prostate cell-specificcontrol sequence; a cancer cell-specific control sequence; and atwo-step transcriptional amplification sequence, wherein the sequencesare operably linked to a polynucleotide encoding a gene producteffective to treat prostate cancer. The polynucleotide construct mayfurther comprise a post-transcriptional control sequence, such as a WPREsequence, and the polynucleotide may be comprised in a liposome.

In a further object of the invention, there is a method of treatingpancreatic cancer in an individual having the cancer, comprisingcontacting at least one pancreatic cancer cell of the individual with atherapeutically effective amount of a polynucleotide constructcomprising a pancreatic cell-specific control sequence and a two-steptranscriptional amplification sequence, both of which are operablylinked to a polynucleotide encoding a gene product effective to treatpancreatic cancer. The construct may further comprise apost-transcriptional control sequence, such as a WPRE sequence. Thepolynucleotide may be comprised in a liposome.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 shows the activity of full-length (FL) topoIIα promoter withluciferase reporter assay in cell lines including normal breastepithelium cell (184A1), normal liver cell (Chang liver), fibroblast(WI38), lung cancer (A549), pancreatic cancer (CAPAN-1), ovarian cancer(iP-1), and breast cancer (MD-MBA-231, MD-MBA-468, MD-MBA-453, SKBR3,T47D, MCF-7) cells.

FIG. 2 demonstrates the activity of full-length (FL) topoIIα promoterwith luciferase reporter assay in cell lines described in FIG. 10.

FIG. 3 shows in vitro luciferase expression between CT572 and CT90 indifferent cell lines by transient transfection, % means compared withthe luciferase activity of CMV-PGL3 vector in individual cell line.

FIG. 4 demonstrates the CT90 promoter activity relative to CMV promoter(CT90/CMV) in tissues of MDA-MB-231 tumor-bearing mice. The promoteractivity in each tissue was determined in luciferase reporter assay asdescribed in the text.

FIG. 5 shows in vitro killing assay of CT90BikDD and CMV-BikDD indifferent cell lines. The Y-axis value indicates the percentage of vitalcells after treatment.

FIG. 6 shows in vivo anti-tumor effect of CT90-BikDD gene therapy.Breast cancer cell line MDA-MB-231 was inoculated 2.5×10⁶ per mouse andmice were treated once per week by liposome-complexed CT0-BikDD,CMV-BikDD, empty vector pGL3, and dextrose buffer D5W as no-treatmentcontrol. Tumor size (Y-axis value) was measured twice per week duringtreatment and showed in the figure. The X-axis indicates the treatmentdates.

FIG. 7 shows promoter activities of TR deletion mutants normalized byCMV promoter activity in each cell line. The percentage in Y-axisindicates the ratio of TfR promoters over CMV promoter in individualcell lines.

FIG. 8 shows promoter activity of CTR116 normalized by CMV promoteractivity in breast cancer cell lines under normoxic and hypoxiccondition. The percentage in Y-axis indicated the ratio of TfR116 overCMV promoter activity.

FIGS. 9A and 9B illustrate constructs of pancreatic specific promoters(FIG. 9A) and their respective luciferase assays (FIG. 9B). The datarepresent mean of four independent experiments; bar, SD.

FIG. 10A illustrates a schematic diagram of CCKAR-based and CMV-basedconstructs, containing the Firefly luciferase reporter gene under thecontrol of the minimal CCKAR promoter without or with WPRE, orCCKAR/TSTA without or with WPRE, or under CMV enhancer/promoter. FIG.10B demonstrates activity of constructs transiently transfected intoAsPC-1, and PANC-1 pancreatic cancer cells. FIG. 10C shows the tissuespecificity of CCKAR-based promoter composites. The data represent meanof four independent experiments; bar, SD.

FIG. 11 shows in vivo transgene expression in orthotopic tumor model ofAsPC cells after systemic delivery of CTP-Luc and CMV-Luc plasmid DNA.FIG. 11A shows in vivo imaging of mice. The representative imaging ofmice are shown. FIG. 11B shows firefly luciferase activity in tissueextracts was quantified with a luminometer and expressed as relativeluciferase units per milligram of total protein. The ratio wascalculated by comparing the level of luciferase activity of CTP mice toCMV mice.

FIG. 12 demonstrates a comparison of firefly luciferase activity underthe control of the CMV enhancer/promoter or hTERTp-based promotercomposites. Firefly luciferase reporter plasmid constructs comprising aCMV enhancer/promoter or hTERTp without or with WPRE, or hTERTp/TSTAwithout or with WPRE were tested in LNCaP cells (FIG. 12A), PC-3prostate cancer cells (FIG. 12B), or WI38 cells (FIG. 12C). The datarepresent mean of four independent experiments; bar, SD.

FIG. 13 shows increased activity of androgen-responsive hTERTp-basedpromoter composites. FIG. 13A shows that an androgen responsive elementARR2 was placed upstream of hTERTp of hTERTp-TSTA-Luc orhTERTp-TSTA-Luc-WPRE composites. ARR2PB-droven Luc was as control. FIG.13B demonstrates activity of hTERTp-based composites in prostate cancercells. The data represent means of four independent experiments; bar,SD.

FIG. 14 provides that the CT90 promoter drives expression of BikDD incomparable level to that from the CMV promoter. MCF7 cells weretransfected with CMV-BikDD and CT90-BikDD by electroporation method.After 24 hours, cells in each experiment were harvested and lysed forWestern blot.

FIG. 15 shows tumor growth during gene therapy treatment. Mice carryingMDA-MB-231 (15A) or MDA-MB-468 (15B) breast cancer xenografts receivedtreatment with 15 μg of lipoplex of CT90-BikDD, CMV-BikDD, empty vectorpGL3, or 5% dextrose in water by intravenous injection. Each treatmentgroup had 10 mice. The mice were treated once a week (QW) for 8 weeks,and the tumor size was measured twice a week.

FIG. 16 provides survival records of MDA-MB-231 breast tumor-bearingmice. The treatment was stopped in the eighth week, and the mice werekept alive until reaching morbid status defined by instituteregulations. The survival number for each week was recorded and shown inthe upper panel. The mean survival time and statistical significancefrom the Kaplan-Meier analysis and log-rank test are shown in the lowerpanel. N.S., not significant.

FIG. 17 demonstrates CT90-BikDD directed selective expression of Bik inbreast cancer cells in vivo. CT90-BikDD or CMV-BikDD lipoplex wasinjected into mice carrying MDA-MB-468 breast cancer xenograft. The micewere sacrificed 72 hours after injection, and tumor and heart wereremoved and fixed. In situ hybridization was performed on the tissuesections to detect BikDD mRNA expression as described in Material andMethods. Shown are representative slides of heart and tumor. In FIG.17A, there is BikDD expression in heart tissues from CMV-BikDD- andCT90-BikDD-treated mice (left and right panels, respectively). Thesamples in upper panels were stained with antisense BikDD probes, andthe deep brown color indicated the positive signals. The lower panelsshowed the negative control experiments stained with sense BikDD probes.In FIG. 17B, there is BikDD expression in tumor tissues from CMV-BikDD-and CT90-BikDD-treated mice (left and right panels, respectively). Thesamples in upper panels were stained with antisense BikDD probes, andthe arrows indicate positive cells. The lower panels showed the negativecontrol experiments stained with sense BikDD probes. In FIG. 17C, theCT90 promoter drives expression of BikDD in comparable level to thatfrom the CMV promoter. MCF7 cells were transfected with CMV-BikDD andCT90-BikDD by electroporation method. After 24 hours, cells in eachexperiment were harvested and lysed for Western blot.

FIG. 18 shows that human cholecystoskinin type-A receptor (CCKAR)promoter is potentially pancreatic cancer-specific. In FIG. 18A, thereare constructs of candidates for pancreatic cancer-specific promoter.CCKAR, orphan G protein-coupled receptor (RDC1), urokinase-typeplasminogen activator receptor (uPAR), and chymotrypsinogen B1 (CTRB1)were polymerase chain reaction-amplified and subcloned into the reporterplasmid pGL3-basic, driving a firefly luciferase gene. In FIG. 18B,PANC-1 and AsPC-1 cells were transiently co-transfected with the plasmidDNA indicated and pRL-TK. Forty-eight hours later, the dual luciferaseratio was measured and shown as relative light units (RLU) normalized tothe Renilla luciferase control. The data represent the mean of fourindependent experiments. Bar, SD.

FIG. 19 demonstrates molecular-engineered cholecystoskinin type-Areceptor (CCKAR)-based promoters are more active and retain pancreaticcancer specificity. In FIG. 19A, there is a schematic diagram ofengineered CCKAR-based constructs including pGL3-CCKAR-Luc-WPRE(CCKAR-P-Luc)), pGL3-CCKAR-TSTA-Luc (CCKAR-T-Luc), andpGL3-CCKAR-TSTA-Luc-WPRE (CTP-Luc). In FIG. 19B, there is activity ofCCKAR-based promoters in pancreatic cancer cells. AsPC-1, PANC-1 andPanO2 cells were transiently co-transfected with plasmid DNA and pRL-TK.Forty-eight hours later, the dual luciferase ratio was measured. Thepercentage relative to the activity of the CMV promoter is shown. Thedata represent the mean of four independent experiments. In FIG. 19C,there is tissue specificity of CCKAR-based promoter composites.Non-pancreatic cancer (LNCaP, PC-3, SKOV3.ip1, MDA-MB-468, and HeLa),and normal and immortalized (WI-38, 184A1, and E6E7) cell lines weretransiently co-transfected with the plasmids indicated and pRL-TK.Forty-eight hours later, the dual luciferase ratio was measured. Thepercentage relative to the activity in AsPC-1 cells is shown. The datarepresent the mean of four independent experiments. RLU, relative lightunits.

FIG. 20 shows the composite of cholecystokinin-type-A receptor(CCKAR)-two-step-transcriptional activation (TSTA)-woodchuck hepatitisvirus posttranscriptional regulatory element (WPRE), CTP, is robust andpancreatic cancer-specific in an orthotopic animal model. Nude micebearing orthotopic AsPC-1 tumors were give 50 μg of DNA in DNA:liposomecomplexes via the tail vein once a day for 3 consecutive days. In FIG.20A, there is in vivo imaging of mice. Mice were anesthetized and imagedfor 5 min using an IVIS imaging system 10 minutes after intraperitonealinjection of D-luciferin. In FIG. 20B, there is tissue distribution ofluciferase expression. Tissue specimens from tumors and organs as shownwere dissected and measured for luciferase activity with a luminometer.Data were expressed as relative luciferase units per milligram of totalprotein. CMV, pGL3-CMV-Luc; CTP, pGL3-CTP-Luc; bar, SD; RLU, relativelight units; n=4 mice each group.

FIG. 21 provides expression of Bik mutant (BikDD) driven by CTP killspancreatic cancer cells effectively and specifically. In FIG. 21A, thereis a schematic diagram of expression constructs in the pUK21 backbone.CMV-BikDD, pUK21-CMV-BikDD; CTP-BikDD, pUK21-CTP-BikDD. In FIG. 21B, thekilling effects of BikDD driven by CMV or CTP are provided. A panel ofpancreatic cancer (AsPC-1, PANC-1, MDA-Panc28, and PanO2), immortalizedhuman pancreatic epithelial E6E7 cells were co-transfected with 2 μg ofpUK21 (negative control), pUK21-CMV-BikDD (positive control), orpUK21-CTP-BikDD, plus 100 ng of pGL3-CMV-Luc. Forty-eight hours aftertransfection, the luciferase activity was imaged for 2 min using an IVISimaging system following a 5-minute incubation with 5 ng/ml ofD-luciferin. Representative images were shown in the upper panel. Thepercentage of the signal compared with the negative control (set as100%) was calculated (lower panel). The data represent the mean of threeindependent experiments. 1, pUK21; 2, CMV-BikDD; 3, CTP-BikDD; bar, SD.

FIG. 22 shows a comparison of firefly luciferase activity under thecontrol of CMV promoter and hTERTp-based promoter composites. In FIG.22A, there is a schematic diagram of reporter constructs. In FIG. 22B,prostate cancer LNCaP and PC-3, and normal human fibroblast WI-38 cellslines were transiently co-transfected with reporter plasmid DNA and theinternal control vector pRL-TK. Forty eight hours later, the dualluciferase ratio was measured. Shown are the luciferase activities(folds) in relative to the CMV promoter (setting at 1).

FIG. 23 demonstrates that a cis-acting ARR2 element further boosts TSTA-and/or WPRE modified hTERTp in response to androgen stimulation. In FIG.23A, there is a schematic diagram of reporter constructs. In FIG. 23B,cells were transiently co-transfected with reporter plasmid DNA and theinternal control vector pRL-TK. Forty eight hours after incubation withR1881, the dual luciferase ratio was measured. Shown are the luciferaseactivities (folds) in relative to that of the CMV promoter (setting at1).

FIG. 24 demonstrates in vivo luciferase expression in LNCaP and PC-3xenografts after systemic delivery of plasmid DNA. Male nude micebearing s.c LNCaP tumors were i.v. injected with DNA (CMV-Luc orATTP-Luc):liposome complexes. Mice were anesthetized, and imaged for 2min with an IVIS™ imaging system following an i.p. injection ofD-luciferin. The imaging shown is 24 h after the last injection. In FIG.24A, mice were then immediately dissected and imaged ex vivo for 2 min.In FIG. 24B, the dissected tumors were then immediately imaged for 10min (FIG. 24C). In FIG. 24D, ex vivo PC-3 tumors were imaged (as C)after systemic delivery of CMV-Luc or ATTP-Luc plasmid DNA to the PC-3tumor-bearing mice. The quantitative signal was presented (right). InFIG. 24E, there is in vivo biodistribution of luciferase expression inthe LNCaP tumor model. Tissue specimens from tumor and organs as shownwere dissected and measured for luciferase activity with a luminometer.

FIG. 25 shows ICBs mediate breast cancer-specific signaling. In FIG.25A, dominant-negative Cdk2 mutant (Cdk2-dn) blocked topoIIα promoteractivity in breast cancer cells. The control vector or Cdk2-dnexpression vector was cotransfected into cells with the topoIIα-pGL3reporter vector, and then luciferase assay was performed to determinetopoIIα promoter activity. The ratio of topoIIα promoter activity in theCdk2-dn group to that in the control group was shown. The cell linesused are described above. BC, breast cancer cells. In FIG. 25B, Cyclin Aactivates the topoIIα promoter in breast cancer cells. The control orcyclin A (CCNA) expression vector was cotransfected into SKBR3 cellswith the topoIIα-pGL3 reporter vector, and then the luciferase assay wasperformed to determine topoIIα promoter activity. The ratio of topoIIαpromoter activity in the Cdk2-dn group to that in the control group isshown. In FIG. 25C, the design of topoIIα promoter deletion mutants(−572, −182, −90, −60) and ICB1-mutated topoIIα promoter (mICB1).Circled x, the mutation at ICB1 site. In FIG. 25D, ICB1 can mediate theactivation of topoIIα promoter by cyclin A/cdk2 signal in breast cancercells. Left panel, The topoIIα promoter deletion mutants werecotransfected with the cyclin A expression vector or the control vectorinto the various cell lines, and their activity was determined byluciferase assay. The ratio of promoter activity in the cyclin A groupto that in the control group (CCNA/Ctrl) is shown. Right panel, The ICB1site of the topoIIα promoter was mutated as described in Materials andMethods. The ICB1-mutated promoter (mICB1-T2A) was cotransfected withthe cyclin A expression vector or the control vector into the variouscell lines, and the activation were determined as described above. InFIG. 25E, the −90 deletion mutant of topoIIα promoter retains breastcancer-specific activity. Upper panel, The T2A90-pGL3 was transfectedinto cell lines, and its promoter activity was determined by using thedual luciferase assay. BE, normal breast epithelial cells; BC, breastcancer cells; LF, normal lung fibroblasts; HC, normal hepatocytes; PC,pancreatic cancer cells; OC, ovarian cancer cells. Lower panel, T2A-90promoter activity relative to that of the CMV promoter in various celllines. The promoter activity ratio of T2A-90 to CMV (topoIIα/CMV) ineach cell line was calculated as described in FIG. 25B.

FIG. 26 demonstrates that enhancer sequence of CMV promoter potentiates−90 topoIIα promoter activity specifically in breast cancer cells. InFIG. 26A, design of CT572, CT182, and CT90 is provided. The CMV promoterenhancer sequence from the pcDNA3.1 vector was PCR amplified and clonedupstream to the topoIIα −182, or −90 promoters in pGL3 reporterconstruct, forming the fusion promoters CT572, CT182, and CT90,respectively. In FIG. 26B, there is activity of CT572, CT182, and CT90in cell lines. The promoter activity in each cell line were determinedas described above. FIG. 26C shows activity of CT572, CT182, and CT90relative to the CMV promoter in cell lines. The promoter activity ratiosof CT572 to CMV (CT572/CMV), CT1 82 to CMV (CT182/CMV), and CT90 to CMV(CT90/CMV) in each cell line were determined as described above. FIG.26D shows CT90 in vivo activity in MDA-MB-231 orthotopic breast cancerxenograft mouse model. The liposome-complexed luciferase reporterconstructs controlled by CT90 (CT90-luc) or CMV promoter (CMV-luc) wereinjected into tumor-bearing mice by tail vein injection. Mice werekilled to remove tumor and major organs 48 hours after injection. Upperpanel, absolute values of luciferase activity from the CMV and CT90promoters in tumor and normal organs. Lower panel, the activity ratio ofthe CT90 promoter to the CMV promoter (CT90/CMV). All values werecalculated as described earlier in FIG. 25B. FIG. 26E shows CT90activity in MDA-MB-231 breast cancer xenograft mice by intratumoralinjection. The CT90-luc or CMV-luc construct complexed with DOTAP:Cholliposome was injected into MDA-MB-231 tumors on mice. Mice were killedto remove tumor and major organs 48 hours after injection. Left panel,absolute values of luciferase activity from the CMV and CT90 promotersin tumor and normal organs. Right panel, the activity ratio of the CT90promoter to the CMV promoter (CT90/CMV). All values were calculated asdescribed earlier in FIG. 25B.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one. Asused herein “another” may mean at least a second or more. In specificembodiments, aspects of the invention may “consist essentially of” oneor more sequences of the invention, for example.

In some embodiments a polynucleotide comprising the inventive controlsequences is delivered by, for example, either a viral or non-viraldelivery system into an appropriate recipient animal to suppress tumorgrowth and development. In one exemplary embodiment of the presentinvention, the delivered therapeutic gene product acts through anapoptosis mechanism to suppress tumor growth and development.

In one aspect of the invention, a therapeutic polypeptide comprised in aconstruct including a tissue-specific control sequence is administeredas a polynucleotide targeted for expression in breast cancer, pancreaticcancer, or prostate cancer, for example. In certain aspects of theinvention, a breast cancer-specific promoter controls expression of thetherapeutic polynucleotide. As used herein, the term “therapeuticpolynucleotide” refers to a polynucleotide that encodes a therapeuticgene product, which may be an RNA, protein, polypeptide, or peptide, forexample.

In a specific embodiment, the control sequences of the present inventioncomprise a composite (chimeric) promoter. For example, breast cancerspecific promoters comprised of a CMV promoter enhancer sequence linkedwith breast cancer specific segments in either topoisomerase IIαpromoter (named as CT90) or transferrin receptor promoter (named asCTR116) may be utilized. Both of these chimeric promoters drive geneexpression selectively in breast cancer cells and possess activitylevels comparable to the CMV promoter. Constructs employing the CT90 orCTR116 chimeric promoters are used in gene transfer to target and treatprimary and metastatic breast cancers with less toxicity to normaltissues, preferably by selectively killing breast cancer cells and/orsignificantly reducing breast tumor growth and/or growth rate.

In other aspects of the invention, a prostate cancer-specific orpancreatic cancer-specific promoter controls expression of a therapeuticpolynucleotide. In a particular embodiment of the invention there is acomposite prostate cancer-specific or pancreatic cancer-specific. Forexample, the prostate cancer-specific promoter may comprise an ARR2control sequence, whereas the pancreatic cancer-specific promoter maycomprise a CCKAR control sequence.

Any promoter or control sequence utilized to regulate expression of atherapeutic polynucleotide may utilize specific regulatory sequencesthat enhance expression and/or post-transcriptional processes, forexample. Particular but exemplary sequences include enhancers, atwo-step transcriptional amplification system, elements that regulateRNA polyadenylation, half-life, and so forth, such as the WPRE, and/orothers in the art.

In other embodiments of the present invention, there are methods ofpreventing growth of a cell in an individual comprising administering tothe individual a construct of the present invention. In specificembodiments, the construct is administered in a liposome and/or thetherapeutic gene product may further comprise a protein transductiondomain (Schwarze et al., 1999), such as HIV Tat or penetratin, forexample. The therapeutic polynucleotide may be administered in a vectorsuch as a plasmid, retroviral vector, adenoviral vector,adeno-associated viral vector, liposome, or a combination thereof, forexample.

I. Nucleic Acid-Based Expression Systems

The present invention utilizes, in some embodiments, systems forexpressing therapeutic polynucleotides, particularly for cancertreatment. Particular exemplary aspects for these polynucleotides aredescribed herein.

A. Vectors

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Maniatis et al., 1988 and Ausubel etal., 1994, both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

1. Promoters and Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.In a specific embodiment, a control sequence, such as a promoter,regulates the tissue specificity within which the nucleic acid sequenceis expressed. A promoter, or control sequence, may comprise geneticelements at which regulatory proteins and molecules may bind, such asRNA polymerase and other transcription factors. The phrases “operativelypositioned,” “operatively linked,” “under control,” and “undertranscriptional control” mean that a promoter or other control sequenceis in a correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence. A promoter may or may not be used inconjunction with an “enhancer,” which refers to a cis-acting regulatorysequence involved in the transcriptional activation of a nucleic acidsequence.

A promoter may be one naturally associated with a gene or sequence, asmay be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other prokaryotic, viral, or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. In addition to producing nucleicacid sequences of promoters and enhancers synthetically, sequences maybe produced using recombinant cloning and/or nucleic acid amplificationtechnology, including PCR™, in connection with the compositionsdisclosed herein (see U.S. Pat. No. 4,683,202; U.S. Pat. No. 5,928,906,each incorporated herein by reference). Furthermore, it is contemplatedthe control sequences that direct transcription and/or expression ofsequences within non-nuclear organelles such as mitochondria,chloroplasts, and the like, can be employed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in the celltype, organelle, and organism chosen for expression. Those of skill inthe art of molecular biology generally know the use of promoters,enhancers, and cell type combinations for protein expression, forexample, see Sambrook et al. (1989), incorporated herein by reference.The promoters employed may be constitutive, tissue-specific, inducible,and/or useful under the appropriate conditions to direct high levelexpression of the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins and/or peptides. Thepromoter may be heterologous or endogenous.

The identity of tissue-specific promoters or elements, as well as assaysto characterize their activity, is well known to those of skill in theart. Tissue-specific promoters utilized to control expression targetingand/or levels of a therapeutic gene product may be comprise wild-typenucleic acid sequence, mutant nucleic acid sequence, or syntheticnucleic acid sequence, so long as the expression of the therapeuticpolynucleotide is preferentially retained in one or more tissues ofinterest compared to tissues that are not the desired target. Controlsequences, such as promoters, may be composite sequences, whereinmultiple regions are derived from different sources. Synthetic controlsequences may be further defined as composite promoters, wherein atleast two separate regions originating from different endogenous and/orsynthetic promoters yet operably linked to control expression of atherapeutic polynucleotide. In a particular embodiment, the tissuespecificity refers to specificity for cancerous tissue, as opposed tonon-cancerous tissue. The term “cancerous tissue” as used herein refersto a tissue comprising at least one cancer cell.

a. Breast Cancer Tissue-Specific Promoter

Most of the promoters currently used in cancer gene therapy possessstrong but unselective activity (e.g. CMV and β-actin promoters) in bothnormal and tumor cells. Thus, in some aspects of the present invention,a breast tissue-specific promoter is utilized in the invention, such asto control expression of a therapeutic polynucleotide, including amutant form of Bik, such as the exemplary BikT33D, BikS35D, and BikT33DS35D mutants. These Bik mutants are described herein but provided infurther detail in U.S. Nonprovisional patent application Ser. No.10/816,698, entitled “Antitumor Effect of Mutant Bik” by Mien-Chie Hung,Yan Li, and Yong Wen, incorporated by reference herein in its entirety.In a particular aspect, the breast cancer-specific promoter of thepresent invention targets expression of a polynucleotide encoding atherapeutic gene product specifically to breast cancer tissue.

In one particular embodiment of the present invention, compositepromoters utilizing either topoisomerase IIα (topoIIα) and transferrinreceptor (TfR) breast cancer-specific control sequences are employed.The topoisomerase IIα (topoIIα) and transferrin receptor (TfR) levelsare elevated in breast cancer, as determined using SAGE analysis andcDNA microarray, for example. The present inventors identified a 90 basepair segment (SEQ ID NO:12) and a 116 base pair segment (SEQ ID NO:13)in the 5′-end of topoIIα and TfR promoter, respectively, as a minimallyrequired breast-cancer specific control sequence. As described in theExamples herein, the promoter activity was enhanced by connecting thesetwo short promoters with an enhancer sequence, such as thecytomegalovirus (CMV) promoter enhancer sequence (SEQ ID NO:11); thesechimeric promoters are referred to herein as CT90 and CTR116,respectively. The full CT90 promoter is comprised in SEQ ID NO:23, andthe full CTR116 promoter is comprised in SEQ ID NO:24.

The CT90 and CTR116 reporter assay in breast cancer cell lines and/or inxenograft mouse models showed that these two promoters possessed notonly strong activity, but also specificity for breast cancer tissue andcells therein. In specific embodiments, the promoter activity of CTR116in cells is further enhanced under hypoxic condition, which usuallyoccurs inside solid tumors. To demonstrate its use in cancer genetherapy, the present inventors generated a DNA construct using CT90 todrive apoptotic gene expression. When transfected into cell lines, thisconstruct selectively killed breast cancer cells. Moreover, the presentinventors demonstrated that this construct had an anti-tumor effect onbreast tumor xenograft in mouse by intravenous injection with anexemplary non-viral delivery system. This indicates that CT90 can drivethe expression of a therapeutic gene, such as mutant Bik, selectively inbreast cancer cells.

Regarding tumor specificity, given that most of the currentcancer-specific promoters have either pretty weak activity compared tothe CMV promoter (e.g. Anderson et al., 2000; Katabi et al., 1999; Lu etal., 2002; Maeda et al., 2001), or insufficient tumor specificity (e.g.CMV-enhanced GAPDH (Qiao et al., 2002), they are not clinically useful.On the contrary, the activities of CT90 and hypoxia-induced CTR116promoters are comparable to CMV promoter (about 0.5- to 2-fold) whilebeing specific for breast cancer cells. Thus, the current inventionencompasses breast cancer-specific promoters for control of expressionof mutant Bik to target breast cancer cells for treatment that is lesstoxic or non-toxic to normal tissues.

b. Pancreatic Cancer Tissue-Specific Promoter

Pancreatic cancer-specific promoters are useful to target pancreaticcancer cells while leaving pancreatic non-cancerous cells unaffected.The present inventors developed strong and pancreatic cancer-specificpromoters for targeted expression of polynucleotides encodingtherapeutic gene products, including mutant Bik, such as the exemplaryBikT33D, BikS35D, and Bik T33DS35D mutants. These Bik mutants aredescribed herein but provided in further detail in U.S. Nonprovisionalpatent application Ser. No. 10/816,698, entitled “Antitumor Effect ofMutant Bik” by Mien-Chie Hung, Yan Li, and Yong Wen, incorporated byreference herein in its entirety.

However, the present inventors developed a pancreatic cancer-specificpromoter as follows. According to literature and the Series Analysis ofGene Expression database of the National Canter for BiotechnologyInformation, the present inventors preliminarily screened a series ofpromoters that target genes overexpressed in human pancreatic cancer,including Cholecystoskinin A receptor (CCKAR), orphan G protein-coupledreceptor (RDC1), urokinase-type plasminogen activator receptor (uPAR),carboxypeptidase A1 (CPA1) and chymotrypsinogen B1 (CTRB1), for example.These were assayed for control of firefly luciferase expression throughluciferase activity in pancreatic cancer cells as well as immortalizednormal cells. The CCKAR promoter ranging from nt −726 to +1 (SEQ IDNO:14) was identified as having optimal activity and specificity amongthese promoters. However, the activity of this minimal CCKAR promoterwas much weaker than that of the commonly used CMV enhancer/promoter.The present inventors then engineered a series of composites based onCCKAR promoter by using the exemplary GAL4-VP 16 or GAL4-VP2 fusionprotein, referred to as a two-step transcriptional amplification (TSTA)system (Iyer et al., 2001; Zhang et al., 2002; Sato et al., 2003; andreferences cited therein), to augment the transcriptional activity; thepost-transcriptional regulatory element of the woodchuck hepatitis virus(WPRE) (SEQ ID NO:15) was utilized to modify RNA polyadenylation signal,RNA export, and/or RNA translation. A skilled artisan recognizes thatthe term “two-step transcriptional amplification (TSTA) system” may alsobe referred to as “two-step transcriptional activation (TSTA) system” or“recombinant transcriptional activation approach” (Nettelbeck et al.,2000). In a particular aspect, the CCKAR-TSTA-WPRE (CTP) promoter isutilized, and an example of such a composite promoter is comprised inSEQ ID NO:20. Thus, the molecularly engineered CTP promoter is employedfor effective treatment modalities for pancreatic cancer gene therapy.

The activity of CCKAR promoter was increased 3.9-fold and 820-fold byWPRE and TSTA, respectively. Surprisingly, for combined TSTA and WPRE,the activity of CCAKAR-TSTA-WPRE (CTP) was 0.7-fold in PANC-1 cells andeven 2.8-fold in AsPC-1 cells compared to CMV promoter, retainingstringent pancreatic cancer specificity. Further, to determine whetherCTP had high activity and strict specificity in vivo after systemicdelivery, nu/nu nude mice bearing subcutaneous (s.c) or orthotopic (o.t)pancreatic tumor of AsPC-1 cells were tail-vein-injected once a day forthree consecutive days with CTP-Luc or CMC-Luc plasmid DNA-DOTOP:Cholcomplexes, and in vivo and ex vivo bioluminescently images with anon-invasive IVIS™ Imaging System were obtained. Bioluminescent imagingshowed very brilliantly in the areas of thorax (lung/heart) in CMV-Lucinjected mice but was almost non-captured in CTP-Luc injected mice. Theactivity of luciferase, detected with a luminometer, demonstrated 1.4-and 2.0-fold greater activity in the o.t. tumors and s.c. tumors,respectively. The ratio of CTP Luciferase expression level to CMVluciferase expression level is 0.37, 0.006, 0.04, 0.37, 0.63, and 0.19in pancreas, lung, heart, liver, spleen, and kidney, respectively, inthe o.t. model, demonstrating improved tissue specificity. Takentogether, molecularly engineered CTP promoter surpasses CMVenhancer/promoter in activity in pancreatic cancer cells and retains itsspecificity in vitro and in vivo, thereby providing safer and moreeffective treatment modalities for pancreatic cancer gene therapy.

c. Prostate Cancer Tissue-Specific Promoter

Prostate cancer-specific promoters can be used to control expression ofpolynucleotides that encode therapeutic gene products, including mutantBik. These Bik mutants are described herein but provided in furtherdetail in U.S. Nonprovisional patent application Ser. No. 10/816,698,entitled “Antitumor Effect of Mutant Bik” by Mien-Chic Hung, Yan Li, andYong Wen, incorporated by reference herein in its entirety. Theactivities of these promoters are androgen-dependent. For numerousdisease stages, patients are androgen-dependent (ADPC), allowing the useof androgen-responsive vectors to direct expression of therapeutic genesto prostatic tissue. Although robust prostate-specific promotersresponsive to androgen receptor have been developed by the presentinventors (Xie et al., Cancer Res 2001) and other groups (Zhang et al.,Mol Endocrinol 2000), these androgen-dependent promoters may be lessactive after castration or androgen ablation therapy, which are the mainmodalities for progressive prostate cancer treatment. Patients treatedwith compositions comprising these promoters may fail this kind oftherapy and die of recurrent androgen-independent prostate cancer(AIPC).

The inventors have developed novel promoters for gene therapy that willbe active in both ADPC and AIPC to treat metastatic and recurrenthormonal refractory prostate cancer. The promoter, referred to herein asATTP, comprises at least a prostate cell-specific control sequence, suchas the exemplary ARR2 regulatory element (SEQ ID NO:17) from ARR2 gene.The promoter may also comprise at least the minimal promoter fragment(hTERTp) of the human telomerase reverse transcriptase (hTERT) (SEQ IDNO:18) operably linked to a two-step transcriptional amplification(TSTA) system, such as the exemplary GAL4-VP16 or GAL4-VP2 (two examplesof GAL4-VP2 are comprised in SEQ ID NO:16 or SEQ ID NO:19) fusionprotein-encoding sequences. The therapeutic polynucleotide may also beoperatively linked to a post-transcriptional control sequence, such asthe post-transcriptional regulatory element of the woodchuck hepatitisvirus (WPRE) to modify RNA polyadenylation signal, RNA export, and/orRNA translation. These regulatory sequences are effective in both ADPCand AIPC cell lines. Given that in most cases of recurrent prostatecancers the AR gene is amplified and/or AR is overexpressed, thisparticular promoter greatly improves the effective index for theembodiment wherein the activity of this system is stimulated byandrogen. In particular embodiments, the TSTA-hTERT-ARR2 and WPREelements are utilized as the prostate cancer-specific control sequences,which in specific embodiments are comprised in SEQ ID NO:21.

Toward the generation of this promoter, the minimal promoter fragment(hTERTp) of the human telomerase reverse transcriptase (hTERT) (SEQ IDNO:18) was PCR-amplified from the DNA extracts of LNCaP cells and testedfor activity in luciferase reporter system. The hTERTp is active in bothLNCaP and PC-3 cells, but its activity was very weak compared to CMVenhancer/promoter. A series of composites based on hTERTp promoter werethen engineered by using the GAL4-VP16 or GAL4-VP2 fusion proteinthrough a two-step transcriptional amplification (TSTA) system toaugment the transcriptional activity and the post-transcriptionalregulatory element of the woodchuck hepatitis virus (WPRE) to modify RNApolyadenylation signal, RNA export, and/or RNA translation. Theexemplary GAL4-VP2 fusion protein is encoded by a polynucleotidecomprising SEQ ID NO:16 or SEQ ID NO:19.

WPRE increased the activity by about 2-fold. Surprisingly, the TSTAsystem can boost the activity up to 67% of CMV activity in LNCaP celland 90% in PC-3 cells. When the TSTA system is utilized in combinationwith WPRE, the activity is comparable to CMV in PC-3, and is even1.5-fold higher in LNCaP cells. In contrast, its activity remains silentin lung fibroblast WI-38 cells. This demonstrated that thehTERTp-TSTA-WPRE system works in both ADPC and AIPC cell lines. In mostcases of recurrent prostate cancers, the AR gene is amplified and/or ARis overexpressed. Therefore, in specific embodiments it greatly improvesthe effective index if the activity of this system can be stimulated byandrogen.

To accomplish this goal, the ARR2 element (SEQ ID NO:17) derived fromplasmid ARR2PB was fused to the hTERTp promoter of phTERTp-TSTA-Luc andphTERTp-TSTA-Luc-WPRE, to produce plasmid pARR2.hTERTp-TSTA-Luc andpARR2.hTERTp-TSTA-Luc-WPRE (ATTP-Luc). As expected, the activity ofARR2.hTERTp-TSTA and ARR2.hTERTp-TSTA-WPRE composites was increased inan androgen-dependent manner, by 15- and 24-fold greater at 10 nm ofandrogen analog R1881, respectively, than that of CMV in LNCaP cells,without there being a significant change in PC-3 cells.

Thus, the present inventors have developed a novel prostate cancerspecific regulatory system that will target polynucleotides that encodetherapeutic gene products to not only ADPC but also AIPC.

2. Initiation Signals and Internal Ribosome Binding Sites

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals. It is well known that theinitiation codon must be “in-frame” with the reading frame of thedesired coding sequence to ensure translation of the entire insert. Theexogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, herein incorporated by reference).

3. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector. (See Carbonelli et al., 1999, Levenson et al., 1998,and Cocea, 1997, incorporated herein by reference.) “Restriction enzymedigestion” refers to catalytic cleavage of a nucleic acid molecule withan enzyme that functions only at specific locations in a nucleic acidmolecule. Many of these restriction enzymes are commercially available.Use of such enzymes is widely understood by those of skill in the art.Frequently, a vector is linearized or fragmented using a restrictionenzyme that cuts within the MCS to enable exogenous sequences to beligated to the vector. “Ligation” refers to the process of formingphosphodiester bonds between two nucleic acid fragments, which may ormay not be contiguous with each other. Techniques involving restrictionenzymes and ligation reactions are well known to those of skill in theart of recombinant technology.

4. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression. (SeeChandler et al., 1997, herein incorporated by reference.)

5. Polyadenylation Signals

In expression, one will typically include a polyadenylation signal toeffect proper polyadenylation of the transcript. The nature of thepolyadenylation signal is not believed to be crucial to the successfulpractice of the invention, and/or any such sequence may be employed.Preferred embodiments include the SV40 polyadenylation signal and/or thebovine growth hormone polyadenylation signal, convenient and/or known tofunction well in various target cells. Also contemplated as an elementof the expression cassette is a transcriptional termination site. Theseelements can serve to enhance message levels and/or to minimize readthrough from the cassette into other sequences.

6. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), which is aspecific nucleic acid sequence at which replication is initiated.Alternatively an autonomously replicating sequence (ARS) can be employedif the host cell is yeast.

7. Selectable and Screenable Markers

In certain embodiments of the invention, the cells contain nucleic acidconstruct of the present invention, a cell may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Generally, aselectable marker is one that confers a property that allows forselection. A positive selectable marker is one in which the presence ofthe marker allows for its selection, while a negative selectable markeris one in which its presence prevents its selection. An example of apositive selectable marker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selectable markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscalorimetric analysis, are also contemplated. Alternatively, screenableenzymes such as herpes simplex virus thymidine kinase (tk) orchloramphenicol acetyltransferase (CAT) may be utilized. One of skill inthe art would also know how to employ immunologic markers, possibly inconjunction with FACS analysis. The marker used is not believed to beimportant, so long as it is capable of being expressed simultaneouslywith the nucleic acid encoding a gene product. Further examples ofselectable and screenable markers are well known to one of skill in theart.

B. Host Cells

The promoters of the present invention may be used in any manner so longas they regulate expression of a particular polynucleotide. Althoughthey are useful for tissue-specific expression, they are by naturepromoters/control sequences and, thus, may be used in any cellenvironment for expressing any polynucleotide.

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these term also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organisms that is capable of replicating a vector and/orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid is transferred or introduced into the host cell.A transformed cell includes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded nucleic acid sequences. Numerous celllines and cultures are available for use as a host cell, and they can beobtained through the American Type Culture Collection (ATCC), which isan organization that serves as an archive for living cultures andgenetic materials. An appropriate host can be determined by one of skillin the art based on the vector backbone and the desired result. Aplasmid or cosmid, for example, can be introduced into a prokaryote hostcell for replication of many vectors. Bacterial cells used as host cellsfor vector replication and/or expression include DH5α, JM109, and KC8,as well as a number of commercially available bacterial hosts such asSURE® Competent Cells and Solopack™ Gold Cells (Stratagene®, La Jolla).Alternatively, bacterial cells such as E. coli LE392 could be used ashost cells for phage viruses.

Examples of eukaryotic host cells for replication and/or expression of avector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12. Manyhost cells from various cell types and organisms are available and wouldbe known to one of skill in the art. Similarly, a viral vector may beused in conjunction with either a eukaryotic or prokaryotic host cell,particularly one that is permissive for replication or expression of thevector.

Some vectors may employ control sequences that allow it to be replicatedand/or expressed in both prokaryotic and eukaryotic cells. One of skillin the art would further understand the conditions under which toincubate all of the above described host cells to maintain them and topermit replication of a vector. Also understood and known are techniquesand conditions that would allow large-scale production of vectors, aswell as production of the nucleic acids encoded by vectors and theircognate polypeptides, proteins, or peptides.

C. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.Although the promoters of the present invention are useful fortissue-specific expression, they are by nature promoters/controlsequences and, thus, may be used in any expression system so long asthey regulate expression of a particular polynucleotide.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MaxBac®2.0 from Invitrogen® and BacPack™ Baculovirus Expression System FromClontech®.

Other examples of expression systems include Stratagene®'s CompleteControl™ Inducible Mammalian Expression System, which involves asynthetic ecdysone-inducible receptor, or its pET Expression System, anE. coli expression system. Another example of an inducible expressionsystem is available from Invitrogen®, which carries the T-Rex™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. Invitrogen®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

II. Nucleic Acid Compositions

In certain embodiments of the present invention, particular sequencesare employed in the inventive polynucleotide constructs and usesthereof. Although a skilled artisan recognizes that these specificsequences may be employed exactly as provided herein, in otherembodiments sequences that are similar to those exemplary sequencesprovided herein are useful at least in part for tissue-specific cancerregulatory sequences.

Certain embodiments of the present invention concern a tissue-specificregulatory nucleic acid (which may interchangeably be used with the term“polynucleotide”). In other aspects, an expression construct nucleicacid comprises a nucleic acid segment of the exemplary SEQ ID NO:12, SEQID NO:13, SEQ ID NO:17, SEQ ID NO:18, or a biologically functionalequivalent thereof.

The term “nucleic acid” is well known in the art. A “nucleic acid” asused herein will generally refer to a molecule (i.e., a strand) of DNA,RNA or a derivative or analog thereof, comprising a nucleobase. Anucleobase includes, for example, a naturally occurring purine orpyrimidine base found in DNA (e.g., an adenine “A,” a guanine “G,” athymine “T” or a cytosine “C”) or RNA (e.g., an A, a G, an uracil “U” ora C). The term “nucleic acid” encompass the terms “oligonucleotide” and“polynucleotide,” each as a subgenus of the term “nucleic acid.” Theterm “oligonucleotide” refers to a molecule of between about 3 and about100 nucleobases in length. The term “polynucleotide” refers to at leastone molecule of greater than about 100 nucleobases in length.

These definitions generally refer to a single-stranded molecule, but inspecific embodiments will also encompass an additional strand that ispartially, substantially or fully complementary to the single-strandedmolecule. Thus, a nucleic acid may encompass a double-stranded moleculeor a triple-stranded molecule that comprises one or more complementarystrand(s) or “complement(s)” of a particular sequence comprising amolecule. As used herein, a single stranded nucleic acid may be denotedby the prefix “ss,” a double stranded nucleic acid by the prefix “ds,”and a triple stranded nucleic acid by the prefix “ts.”

1. Nucleobases

As used herein a “nucleobase” refers to a heterocyclic base, such as forexample a naturally occurring nucleobase (i.e., an A, T, G, C or U)found in at least one naturally occurring nucleic acid (i.e., DNA andRNA), and naturally or non-naturally occurring derivative(s) and analogsof such a nucleobase. A nucleobase generally can form one or morehydrogen bonds (“anneal” or “hybridize”) with at least one naturallyoccurring nucleobase in manner that may substitute for naturallyoccurring nucleobase pairing (e.g., the hydrogen bonding between A andT, G and C, and A and U).

“Purine” and/or “pyrimidine” nucleobase(s) encompass naturally occurringpurine and/or pyrimidine nucleobases and also derivative(s) andanalog(s) thereof, including but not limited to, those a purine orpyrimidine substituted by one or more of an alkyl, caboxyalkyl, amino,hydroxyl, halogen (i.e., fluoro, chloro, bromo, or iodo), thiol oralkylthiol moeity. Preferred alkyl (e.g., alkyl, caboxyalkyl, etc.)moeities comprise of from about 1, about 2, about 3, about 4, about 5,to about 6 carbon atoms. Other non-limiting examples of a purine orpyrimidine include a deazapurine, a 2,6-diaminopurine, a 5-fluorouracil,a xanthine, a hypoxanthine, a 8-bromoguanine, a 8-chloroguanine, abromothymine, a 8-aminoguanine, a 8-hydroxyguanine, a 8-methylguanine, a8-thioguanine, an azaguanine, a 2-aminopurine, a 5-ethylcytosine, a5-methylcyosine, a 5-bromouracil, a 5-ethyluracil, a 5-iodouracil, a5-chlorouracil, a 5-propyluracil, a thiouracil, a 2-methyladenine, amethylthioadenine, a N,N-diemethyladenine, an azaadenines, a8-bromoadenine, a 8-hydroxyadenine, a 6-hydroxyaminopurine, a6-thiopurine, a 4-(6-aminohexyl/cytosine), and the like.

A nucleobase may be comprised in a nucleoside or nucleotide, using anychemical or natural synthesis method described herein or known to one ofordinary skill in the art.

2. Nucleosides

As used herein, a “nucleoside” refers to an individual chemical unitcomprising a nucleobase covalently attached to a nucleobase linkermoiety. A non-limiting example of a “nucleobase linker moiety” is asugar comprising 5-carbon atoms (i.e., a “5-carbon sugar”), includingbut not limited to a deoxyribose, a ribose, an arabinose, or aderivative or an analog of a 5-carbon sugar. Non-limiting examples of aderivative or an analog of a 5-carbon sugar include a2′-fluoro-2′-deoxyribose or a carbocyclic sugar where a carbon issubstituted for an oxygen atom in the sugar ring.

Different types of covalent attachment(s) of a nucleobase to anucleobase linker moiety are known in the art. By way of non-limitingexample, a nucleoside comprising a purine (i.e., A or G) or a7-deazapurine nucleobase typically covalently attaches the 9 position ofa purine or a 7-deazapurine to the 1′-position of a 5-carbon sugar. Inanother non-limiting example, a nucleoside comprising a pyrimidinenucleobase (i.e., C, T or U) typically covalently attaches a 1 positionof a pyrimidine to a 1′-position of a 5-carbon sugar (Kornberg andBaker, 1992).

3. Nucleotides

As used herein, a “nucleotide” refers to a nucleoside further comprisinga “backbone moiety”. A backbone moiety generally covalently attaches anucleotide to another molecule comprising a nucleotide, or to anothernucleotide to form a nucleic acid. The “backbone moiety” in naturallyoccurring nucleotides typically comprises a phosphorus moiety, which iscovalently attached to a 5-carbon sugar. The attachment of the backbonemoiety typically occurs at either the 3′- or 5′-position of the 5-carbonsugar. However, other types of attachments are known in the art,particularly when a nucleotide comprises derivatives or analogs of anaturally occurring 5-carbon sugar or phosphorus moiety.

4. Nucleic Acid Analogs

A nucleic acid may comprise, or be composed entirely of, a derivative oranalog of a nucleobase, a nucleobase linker moiety and/or backbonemoiety that may be present in a naturally occurring nucleic acid. Asused herein a “derivative” refers to a chemically modified or alteredform of a naturally occurring molecule, while the terms “mimic” or“analog” refer to a molecule that may or may not structurally resemble anaturally occurring molecule or moiety, but possesses similar functions.As used herein, a “moiety” generally refers to a smaller chemical ormolecular component of a larger chemical or molecular structure.Nucleobase, nucleoside and nucleotide analogs or derivatives are wellknown in the art, and have been described (see for example, Scheit,1980, incorporated herein by reference).

Additional non-limiting examples of nucleosides, nucleotides or nucleicacids comprising 5-carbon sugar and/or backbone moiety derivatives oranalogs, include those in U.S. Pat. No. 5,681,947 which describesoligonucleotides comprising purine derivatives that form triple helixeswith and/or prevent expression of dsDNA; U.S. Pat. Nos. 5,652,099 and5,763,167 which describe nucleic acids incorporating fluorescent analogsof nucleosides found in DNA or RNA, particularly for use as fluorescentnucleic acids probes; U.S. Pat. No. 5,614,617 which describesoligonucleotide analogs with substitutions on pyrimidine rings thatpossess enhanced nuclease stability; U.S. Pat. Nos. 5,670,663, 5,872,232and 5,859,221 which describe oligonucleotide analogs with modified5-carbon sugars (i.e., modified 2′-deoxyfuranosyl moieties) used innucleic acid detection; U.S. Pat. No. 5,446,137 which describesoligonucleotides comprising at least one 5-carbon sugar moietysubstituted at the 4′ position with a substituent other than hydrogenthat can be used in hybridization assays; U.S. Pat. No. 5,886,165 whichdescribes oligonucleotides with both deoxyribonucleotides with 3′-5′internucleotide linkages and ribonucleotides with 2′-5′ internucleotidelinkages; U.S. Pat. No. 5,714,606 which describes a modifiedinternucleotide linkage wherein a 3′-position oxygen of theinternucleotide linkage is replaced by a carbon to enhance the nucleaseresistance of nucleic acids; U.S. Pat. No. 5,672,697 which describesoligonucleotides containing one or more 5′ methylene phosphonateinternucleotide linkages that enhance nuclease resistance; U.S. Pat.Nos. 5,466,786 and 5,792,847 which describe the linkage of a substituentmoeity which may comprise a drug or label to the 2′ carbon of anoligonucleotide to provide enhanced nuclease stability and ability todeliver drugs or detection moieties; U.S. Pat. No. 5,223,618 whichdescribes oligonucleotide analogs with a 2 or 3 carbon backbone linkageattaching the 4′ position and 3′ position of adjacent 5-carbon sugarmoiety to enhanced cellular uptake, resistance to nucleases andhybridization to target RNA; U.S. Pat. No. 5,470,967 which describesoligonucleotides comprising at least one sulfamate or sulfamideinternucleotide linkage that are useful as nucleic acid hybridizationprobe; U.S. Pat. Nos. 5,378,825, 5,777,092, 5,623,070, 5,610,289 and5,602,240 which describe oligonucleotides with three or four atom linkermoeity replacing phosphodiester backbone moeity used for improvednuclease resistance, cellular uptake and regulating RNA expression; U.S.Pat. No. 5,858,988 which describes hydrophobic carrier agent attached tothe 2′-O position of oligonuceotides to enhanced their membranepermeability and stability; U.S. Pat. No. 5,214,136 which describesoligonucleotides conjugaged to anthraquinone at the 5′ terminus thatpossess enhanced hybridization to DNA or RNA; enhanced stability tonucleases; U.S. Pat. No. 5,700,922 which describes PNA-DNA-PNA chimeraswherein the DNA comprises 2′-deoxy-erythro-pentofuranosyl nucleotidesfor enhanced nuclease resistance, binding affinity, and ability toactivate RNase H; and U.S. Pat. No. 5,708,154 which describes RNA linkedto a DNA to form a DNA-RNA hybrid.

5. Preparation of Nucleic Acids

A nucleic acid may be made by any technique known to one of ordinaryskill in the art, such as for example, chemical synthesis, enzymaticproduction or biological production. Non-limiting examples of asynthetic nucleic acid (e.g., a synthetic oligonucleotide), include anucleic acid made by in vitro chemically synthesis usingphosphotriester, phosphite or phosphoramidite chemistry and solid phasetechniques such as described in EP 266,032, incorporated herein byreference, or via deoxynucleoside H-phosphonate intermediates asdescribed by Froehler et al., 1986 and U.S. Pat. No. 5,705,629, eachincorporated herein by reference. In the methods of the presentinvention, one or more oligonucleotide may be used. Various differentmechanisms of oligonucleotide synthesis have been disclosed in forexample, U.S. Pat. Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566,4,959,463, 5,428,148, 5,554,744, 5,574,146, 5,602,244, each of which isincorporated herein by reference.

A non-limiting example of an enzymatically produced nucleic acid includeone produced by enzymes in amplification reactions such as PCRTM (seefor example, U.S. Pat. No. 4,683,202 and U.S. Pat. No. 4,682,195, eachincorporated herein by reference), or the synthesis of anoligonucleotide described in U.S. Pat. No. 5,645,897, incorporatedherein by reference. A non-limiting example of a biologically producednucleic acid includes a recombinant nucleic acid produced (i.e.,replicated) in a living cell, such as a recombinant DNA vectorreplicated in bacteria (see for example, Sambrook et al. 1989,incorporated herein by reference).

6. Purification of Nucleic Acids

A nucleic acid may be purified on polyacrylamide gels, cesium chloridecentrifugation gradients, or by any other means known to one of ordinaryskill in the art (see for example, Sambrook et al., 1989, incorporatedherein by reference).

In certain aspect, the present invention concerns a nucleic acid that isan isolated nucleic acid. As used herein, the term “isolated nucleicacid” refers to a nucleic acid molecule (e.g., an RNA or DNA molecule)that has been isolated free of, or is otherwise free of, the bulk of thetotal genomic and transcribed nucleic acids of one or more cells. Incertain embodiments, “isolated nucleic acid” refers to a nucleic acidthat has been isolated free of, or is otherwise free of, bulk ofcellular components or in vitro reaction components such as for example,macromolecules such as lipids or proteins, small biological molecules,and the like.

7. Nucleic Acid Segments

In certain embodiments, the nucleic acid is a nucleic acid segment. Asused herein, the term “nucleic acid segment,” are smaller fragments of anucleic acid, such as for non-limiting example, those that comprise onlypart of the regulatory sequences for a given transcribed polynucleotide.

8. Nucleic Acid Complements

The present invention also encompasses a nucleic acid that iscomplementary to a nucleic acid of the invention. In particularembodiments the invention encompasses a nucleic acid or a nucleic acidsegment complementary to the sequence set forth in SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:17, and SEQ ID NO:18, for example. A nucleic acid is“complement(s)” or is “complementary” to another nucleic acid when it iscapable of base-pairing with another nucleic acid according to thestandard Watson-Crick, Hoogsteen or reverse Hoogsteen bindingcomplementarity rules. As used herein “another nucleic acid” may referto a separate molecule or a spatial separated sequence of the samemolecule.

As used herein, the term “complementary” or “complement(s)” also refersto a nucleic acid comprising a sequence of consecutive nucleobases orsemiconsecutive nucleobases (e.g., one or more nucleobase moieties arenot present in the molecule) capable of hybridizing to another nucleicacid strand or duplex even if less than all the nucleobases do not basepair with a counterpart nucleobase. In certain embodiments, a“complementary” nucleic acid comprises a sequence in which about 70%,about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about77%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, to about 100%, and any rangederivable therein, of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization. In certain embodiments, the term “complementary” refersto a nucleic acid that may hybridize to another nucleic acid strand orduplex in stringent conditions, as would be understood by one ofordinary skill in the art.

In certain embodiments, a “partly complementary” nucleic acid comprisesa sequence that may hybridize in low stringency conditions to a singleor double stranded nucleic acid, or contains a sequence in which lessthan about 70% of the nucleobase sequence is capable of base-pairingwith a single or double stranded nucleic acid molecule duringhybridization.

9. Hybridization

As used herein, “hybridization”, “hybridizes” or “capable ofhybridizing” is understood to mean the forming of a double or triplestranded molecule or a molecule with partial double or triple strandednature. The term “anneal” as used herein is synonymous with “hybridize.”The term “hybridization”, “hybridize(s)” or “capable of hybridizing”encompasses the terms “stringent condition(s)” or “high stringency” andthe terms “low stringency” or “low stringency condition(s).”

As used herein “stringent condition(s)” or “high stringency” are thoseconditions that allow hybridization between or within one or morenucleic acid strand(s) containing complementary sequence(s), butprecludes hybridization of random sequences. Stringent conditionstolerate little, if any, mismatch between a nucleic acid and a targetstrand. Such conditions are well known to those of ordinary skill in theart, and are preferred for applications requiring high selectivity.Non-limiting applications include isolating a nucleic acid, such as agene or a nucleic acid segment thereof, or detecting at least onespecific mRNA transcript or a nucleic acid segment thereof, and thelike.

Stringent conditions may comprise low salt and/or high temperatureconditions, such as provided by about 0.02 M to about 0.15 M NaCl attemperatures of about 50° C. to about 70° C. It is understood that thetemperature and ionic strength of a desired stringency are determined inpart by the length of the particular nucleic acid(s), the length andnucleobase content of the target sequence(s), the charge composition ofthe nucleic acid(s), and to the presence or concentration of formamide,tetramethylammonium chloride or other solvent(s) in a hybridizationmixture.

It is also understood that these ranges, compositions and conditions forhybridization are mentioned by way of non-limiting examples only, andthat the desired stringency for a particular hybridization reaction isoften determined empirically by comparison to one or more positive ornegative controls. Depending on the application envisioned it ispreferred to employ varying conditions of hybridization to achievevarying degrees of selectivity of a nucleic acid towards a targetsequence. In a non-limiting example, identification or isolation of arelated target nucleic acid that does not hybridize to a nucleic acidunder stringent conditions may be achieved by hybridization at lowtemperature and/or high ionic strength. Such conditions are termed “lowstringency” or “low stringency conditions”, and non-limiting examples oflow stringency include hybridization performed at about 0.15 M to about0.9 M NaCl at a temperature range of about 20° C. to about 50° C. Ofcourse, it is within the skill of one in the art to further modify thelow or high stringency conditions to suite a particular application.

The nucleic acid(s) of the present invention, regardless of the lengthof the sequence itself, may be combined with other nucleic acidsequences, including but not limited to, promoters, enhancers,polyadenylation signals, restriction enzyme sites, multiple cloningsites, coding segments, and the like, to create one or more nucleic acidconstruct(s). As used herein, a “nucleic acid construct” is a nucleicacid engineered or altered by the hand of man, and generally comprisesone or more nucleic acid sequences organized by the hand of man.

In a non-limiting example, one or more nucleic acid constructs may beprepared that include a contiguous stretch of nucleotides identical toor complementary to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, or SEQ IDNO:18, for example. A nucleic acid construct may be about 3, about 5,about 8, about 10 to about 14, or about 15, about 20, about 30, about40, about 50, about 100, about 200, about 500, about 1,000, about 2,000,about 3,000, about 5,000, about 10,000, about 15,000, about 20,000,about 30,000, about 50,000, about 100,000, about 250,000, about 500,000,about 750,000, to about 1,000,000 nucleotides in length, as well asconstructs of greater size, up to and including chromosomal sizes(including all intermediate lengths and intermediate ranges), given theadvent of nucleic acids constructs such as a yeast artificial chromosomeare known to those of ordinary skill in the art. It will be readilyunderstood that “intermediate lengths” and “intermediate ranges”, asused herein, means any length or range including or between the quotedvalues (i.e., all integers including and between such values).Non-limiting examples of intermediate lengths include about 11, about12, about 13, about 16, about 17, about 18, about 19, etc.; about 21,about 22, about 23, etc.; about 31, about 32, etc.; about 51, about 52,about 53, etc.; about 101, about 102, about 103, etc.; about 151, about152, about 153, etc.; about 1,001, about 1002, etc; about 50,001, about50,002, etc; about 750,001, about 750,002, etc.; about 1,000,001, about1,000,002, etc. Non-limiting examples of intermediate ranges includeabout 3 to about 32, about 150 to about 500,001, about 3,032 to about7,145, about 5,000 to about 15,000, about 20,007 to about 1,000,003,etc.

The term “a sequence essentially as set forth in SEQ ID NO:12” or “asequence essentially as set forth in SEQ ID NO:13”, for example, meansthat the sequence substantially corresponds to a portion of SEQ ID NO:12and SEQ ID NO:13 and has relatively few nucleotides that are notidentical to, or a biologically functional equivalent of, the respectivenucleotides of SEQ ID NO:12 and/or SEQ ID NO:13. Thus, “a sequenceessentially as set forth in SEQ ID NO:12” or “a sequence essentially asset forth in SEQ ID NO:13” encompasses nucleic acids, nucleic acidsegments, and genes that comprise part or all of the nucleic acidsequences as set forth in SEQ ID NO:12 and/or SEQ ID NO:13.

The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein. Accordingly, a sequencethat has between about 70% and about 80%; or more preferably, betweenabout 81% and about 90%; or even more preferably, between about 91% andabout 99%; of nucleotides that are identical or functionally equivalentto the nucleotides of sequences referred to herein, such as theexemplary SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17 or SEQ ID NO:18 willbe a sequence that is respectively “essentially as set forth in the SEQID NO:12, SEQ ID NO:13, SEQ ID NO:17 or SEQ ID NO:18”, provided thebiological activity of the sequences is maintained.

In certain other embodiments, the invention concerns at least onerecombinant vector that include within its sequence a nucleic acidsequence essentially as set forth in SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:17, or SEQ ID NO:18.

III. Therapeutic Polynucleotides

The therapeutic polynucleotide which expression is controlled by theinventive control sequences encompassed by the invention may be of anykind, so long as the gene product encoded thereby generates ananticancer effect. Anticancer effects include inducing apoptosis in atleast one cancer cell, inhibiting proliferation of at least one cancercell, ameliorating at least once symptom of cancer in an individual, andso forth. In particular embodiments, the therapeutic polynucleotideencodes a mutant form of Bik, including the exemplary BikT33D, BikS35D,and Bik T33DS35D mutants, which are described in U.S. patent applicationSer. No. 10/816,698, incorporated by reference herein in its entirety.

The therapeutic polynucleotide may be of any kind known to those ofskill in the art or discovered later. In particular embodiments, theyencode inhibitors of cellular proliferation, regulators of programmedcell death, tumor suppressors and/or antisense sequences of inducers ofcellular proliferation. The therapeutic polynucleotide may encode smallinterfering RNAs or antisense sequences. Examples of therapeuticpolynucleotides include those encoding TNFα or p53 or that encodepolypeptide inducers of apoptosis including, but not limited to, Bik,p53, Bax, Bak, Bcl-x, Bad, Bim, Bok, Bid, Harakiri, Ad E1B, Bad andICE-CED3 proteases. Other exemplary therapeutic polynucleotides includethose that encode retinoblastoma, Blk, IL-12, IL-10, IFN-a, cytosinedeaminase, GM-CSF, E1A, and other pro-apoptotic proteins, for example. Apolynucleotide encoding an amino acid substitution at threonine 33,serine 35, or both of mutant Bik may be utilized. In particular aspectsof these embodiments, the amino acids of the mutant Bik polypeptide aresubstituted with aspartate. In other particular aspects, one or morephosphorylation sites are defective in a mutant Bik. Additionaltherapeutic polynucleotides include TNFα or p53 or inducers of apoptosisincluding, but not limited to, Bik, p53, Bax, Bak, Bcl-x, Bad, Bim, Bok,Bid, Harakiri, Ad E1B, Bad and ICE-CED3 proteases.

IV. Nucleic Acid Delivery

The general approach to the aspects of the present invention concerningcompositions and/or therapeutics is to provide a cell with a geneconstruct encoding a specific and/or desired mutant Bik protein,polypeptide, or peptide, thereby permitting the desired activity of theprotein, polypeptide, or peptide to take effect. While it is conceivablethat the gene construct and/or protein may be delivered directly, apreferred embodiment involves providing a nucleic acid encoding aspecific and desired protein, polypeptide, or peptide to the cell.Following this provision, the proteinaceous composition is synthesizedby the transcriptional and translational machinery of the cell, as wellas any that may be provided by the expression construct. In providingantisense, ribozymes and other inhibitors, the preferred mode is also toprovide a nucleic acid encoding the construct to the cell.

In certain embodiments of the invention, the nucleic acid encoding thegene may be stably integrated into the genome of the cell. In yetfurther embodiments, the nucleic acid may be stably maintained in thecell as a separate, episomal segment of DNA. Such nucleic acid segmentsand “episomes” encode sequences sufficient to permit maintenance andreplication independent of and in synchronization with the host cellcycle. How the expression construct is delivered to a cell and/or wherein the cell the nucleic acid remains is dependent on the type ofexpression construct employed.

A. DNA Delivery Using Viral Vectors

The ability of certain viruses to infect cells and enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand/or express viral genes stably and/or efficiently have made themattractive candidates for the transfer of foreign genes into mammaliancells. Preferred gene therapy vectors of the present invention willgenerally be viral vectors.

Although some viruses that can accept foreign genetic material arelimited in the number of nucleotides they can accommodate and/or in therange of cells they infect, these viruses have been demonstrated tosuccessfully effect gene expression. However, adenoviruses do notintegrate their genetic material into the host genome and/or thereforedo not require host replication for gene expression, making them ideallysuited for rapid, efficient, heterologous gene expression. Techniquesfor preparing replication-defective infective viruses are well known inthe art.

Of course, in using viral delivery systems, one will desire to purifythe virion sufficiently to render it essentially free of undesirablecontaminants, such as defective interfering viral particles andendotoxins and other pyrogens such that it will not cause any untowardreactions in the cell, animal and/or individual receiving the vectorconstruct. A preferred means of purifying the vector involves the use ofbuoyant density gradients, such as cesium chloride gradientcentrifugation.

1. Adenoviral Vectors

A particular method for delivery of the expression constructs involvesthe use of an adenovirus expression vector. Although adenovirus vectorsare known to have a low capacity for integration into genomic DNA, thisfeature is counterbalanced by the high efficiency of gene transferafforded by these vectors. “Adenovirus expression vector” is meant toinclude those constructs containing adenovirus sequences sufficient to(a) support packaging of the construct and/or (b) to ultimately expressa tissue and/or cell-specific construct that has been cloned therein.

The expression vector comprises a genetically engineered form ofadenovirus. Knowledge of the genetic organization and adenovirus, a 36kb, linear, double-stranded DNA virus, allows substitution of largepieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus andHorwitz, 1992). In contrast to retrovirus, the adenoviral infection ofhost cells does not result in chromosomal integration because adenoviralDNA can replicate in an episomal manner without potential genotoxicity.Also, adenoviruses are structurally stable, and/or no genomerearrangement has been detected after extensive amplification.

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized genome, ease of manipulation, high titer, widetarget-cell range and/or high infectivity. Both ends of the viral genomecontain 100-200 base pair inverted repeats (ITRs), which are ciselements necessary for viral DNA replication and/or packaging. The early(E) and/or late (L) regions of the genome contain differenttranscription units that are divided by the onset of viral DNAreplication. The E1 region (E1A and/or E1B) encodes proteins responsiblefor the regulation of transcription of the viral genome and/or a fewcellular genes. The expression of the E2 region (E2A and/or E2B) resultsin the synthesis of the proteins for viral DNA replication. Theseproteins are involved in DNA replication, late gene expression and/orhost cell shut-off (Renan, 1990). The products of the late genes,including the majority of the viral capsid proteins, are expressed onlyafter significant processing of a single primary transcript issued bythe major late promoter (MLP). The MLP (located at 16.8 m.u.) isparticularly efficient during the late phase of infection, and/or allthe mRNA's issued from this promoter possess a 5′-tripartite leader(TPL) sequence which makes them preferred mRNA's for translation.

In a current system, recombinant adenovirus is generated from homologousrecombination between shuttle vector and provirus vector. Due to thepossible recombination between two proviral vectors, wild-typeadenovirus may be generated from this process. Therefore, it is criticalto isolate a single clone of virus from an individual plaque and/orexamine its genomic structure.

Generation and/or propagation of the current adenovirus vectors, whichare replication deficient, depend on a unique helper cell line,designated 293, which was transformed from human embryonic kidney cellsby Ad5 DNA fragments and constitutively expresses E1 proteins (E1Aand/or E1B; Graham et al., 1977). Since the E3 region is dispensablefrom the adenovirus genome (Jones and Shenk, 1978), the currentadenovirus vectors, with the help of 293 cells, carry foreign DNA ineither the E1, the D3 and both regions (Graham and Prevec, 1991).Recently, adenoviral vectors comprising deletions in the E4 region havebeen described (U.S. Pat. No. 5,670,488, incorporated herein byreference).

In nature, adenovirus can package approximately 105% of the wild-typegenome (Ghosh-Choudhury et al., 1987), providing capacity for about 2extra kb of DNA. Combined with the approximately 5.5 kb of DNA that isreplaceable in the E1 and/or E3 regions, the maximum capacity of thecurrent adenovirus vector is under 7.5 kb, and/or about 15% of the totallength of the vector. More than 80% of the adenovirus viral genomeremains in the vector backbone.

Helper cell lines may be derived from human cells such as humanembryonic kidney cells, muscle cells, hematopoietic cells and otherhuman embryonic mesenchymal and epithelial cells. Alternatively, thehelper cells may be derived from the cells of other mammalian speciesthat are permissive for human adenovirus. Such cells include, e.g., Verocells and other monkey embryonic mesenchymal and/or epithelial cells. Asstated above, the preferred helper cell line is 293.

Recently, Racher et al. (1995) disclosed improved methods for culturing293 cells and/or propagating adenovirus. In one format, natural cellaggregates are grown by inoculating individual cells into 1 litersiliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 mlof medium. Following stirring at 40 rpm, the cell viability is estimatedwith trypan blue. In another format, Fibra-Cel microcaniers (BibbySterlin, Stone, UK) (5 g/l) is employed as follows. A cell inoculum,resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250ml Erlenmeyer flask and/or left stationary, with occasional agitation,for 1 to 4 h. The medium is then replaced with 50 ml of fresh mediumand/or shaking initiated. For virus production, cells are allowed togrow to about 80% confluence, after which time the medium is replaced(to 25% of the final volume) and/or adenovirus added at an MOI of 0.05.Cultures are left stationary overnight, following which the volume isincreased to 100% and/or shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replicationdefective, and at least conditionally defective, the nature of theadenovirus vector is not believed to be crucial to the successfulpractice of the invention. The adenovirus may be of any of the 42different known serotypes and subgroups A-F. Adenovirus type 5 ofsubgroup C is the preferred starting material in order to obtain theconditional replication-defective adenovirus vector for use in thepresent invention. This is because Adenovirus type 5 is a humanadenovirus about which a great deal of biochemical and geneticinformation is known, and it has historically been used for mostconstructions employing adenovirus as a vector.

As stated above, the typical vector according to the present inventionis replication defective and will not have an adenovirus E1 region.Thus, it will be most convenient to introduce the transforming constructat the position from which the E1-coding sequences have been removed.However, the position of insertion of the construct within theadenovirus sequences is not critical to the invention. Thepolynucleotide encoding the gene of interest may also be inserted inlieu of the deleted E3 region in E3 replacement vectors as described byKarlsson et al. (1986) and in the E4 region where a helper cell line andhelper virus complements the E4 defect.

Adenovirus growth and/or manipulation is known to those of skill in theart, and/or exhibits broad host range in vitro and in vivo. This groupof viruses can be obtained in high titers, e.g., 10⁹ to 10¹¹plaque-forming units per ml, and they are highly infective. The lifecycle of adenovirus does not require integration into the host cellgenome. The foreign genes delivered by adenovirus vectors are episomaland, therefore, have low genotoxicity to host cells. No side effectshave been reported in studies of vaccination with wild-type adenovirus(Couch et al., 1963; Top et al., 1971), demonstrating their safetyand/or therapeutic potential as in vivo gene transfer vectors.

Adenovirus vectors have been used in eukaryotic gene expression (Levreroet al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhausand Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studiessuggested that recombinant adenovirus could be used for gene therapy(Stratford-Perricaudet and Perricaudet, 1991a; Stratford-Perricaudet etal., 1991b; Rich et al., 1993). Studies in administering recombinantadenovirus to different tissues include trachea instillation (Rosenfeldet al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,1993), peripheral intravenous injections (Herz and Gerard, 1993) and/orstereotactic inoculation into the brain (Le Gal La Salle et al., 1993).Recombinant adenovirus and adeno-associated virus (see below) can bothinfect and transduce non-dividing human primary cells.

2. AAV Vectors

Adeno-associated virus (AAV) is an attractive vector system for use inthe cell transduction of the present invention as it has a highfrequency of integration and it can infect nondividing cells, thusmaking it useful for delivery of genes into mammalian cells, forexample, in tissue culture (Muzyczka, 1992) and in vivo. AAV has a broadhost range for infectivity (Tratschin et al., 1984; Laughlin et al.,1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Detailsconcerning the generation and use of rAAV vectors are described in U.S.Pat. No. 5,139,941 and/or U.S. Pat. No. 4,797,368, each incorporatedherein by reference.

Studies demonstrating the use of AAV in gene delivery include LaFace etal. (1988); Zhou et al. (1993); Flotte et al. (1993); and Walsh et al.(1994). Recombinant AAV vectors have been used successfully for in vitroand/or in vivo transduction of marker genes (Kaplitt et al., 1994;Lebkowski et al., 1988; Samulski et al., 1989; Yoder et al., 1994; Zhouet al., 1994; Hermonat and Muzyczka, 1984; Tratschin et al., 1985;McLaughlin et al., 1988) and genes involved in human diseases (Flotte etal., 1992; Luo et al., 1994; Ohi et al., 1990; Walsh et al., 1994; Weiet al., 1994). Recently, an AAV vector has been approved for phase Ihuman trials for the treatment of cystic fibrosis.

AAV is a dependent parvovirus in that it requires coinfection withanother virus (either adenovirus and a member of the herpes virusfamily) to undergo a productive infection in cultured cells (Muzyczka,1992). In the absence of coinfection with helper virus, the wild typeAAV genome integrates through its ends into human chromosome 19 where itresides in a latent state as a provirus (Kotin et al., 1990; Samulski etal., 1991). rAAV, however, is not restricted to chromosome 19 forintegration unless the AAV Rep protein is also expressed (Shelling andSmith, 1994). When a cell carrying an AAV provirus is superinfected witha helper virus, the AAV genome is “rescued” from the chromosome and froma recombinant plasmid, and/or a normal productive infection isestablished (Samulski et al., 1989; McLaughlin et al., 1988; Kotin etal., 1990; Muzyczka, 1992).

Typically, recombinant AAV (rAAV) virus is made by cotransfecting aplasmid containing the gene of interest flanked by the two AAV terminalrepeats (McLaughlin et al., 1988; Samulski et al., 1989; eachincorporated herein by reference) and/or an expression plasmidcontaining the wild type AAV coding sequences without the terminalrepeats, for example pIM45 (McCarty et al., 1991; incorporated herein byreference). The cells are also infected and transfected with adenovirusand plasmids carrying the adenovirus genes required for AAV helperfunction. rAAV virus stocks made in such fashion are contaminated withadenovirus which must be physically separated from the rAAV particles(for example, by cesium chloride density centrifugation). Alternatively,adenovirus vectors containing the AAV coding regions and cell linescontaining the AAV coding regions and some and all of the adenovirushelper genes could be used (Yang et al., 1994; Clark et al., 1995). Celllines carrying the rAAV DNA as an integrated provirus can also be used(Flotte et al., 1995).

3. Retroviral Vectors

Retroviruses have promise as gene delivery vectors due to their abilityto integrate their genes into the host genome, transferring a largeamount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines(Miller, 1992).

The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and/or directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and/or its descendants. The retroviral genome contains three genes,gag, pol, and/or env that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene contains a signal for packaging of the genome into virions.Two long terminal repeat (LTR) sequences are present at the 5′ and 3′ends of the viral genome. These contain strong promoter and enhancersequences and are also required for integration in the host cell genome(Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding agene of interest is inserted into the viral genome in the place ofcertain viral sequences to produce a virus that isreplication-defective. In order to produce virions, a packaging cellline containing the gag, pol, and env genes but without the LTR andpackaging components is constructed (Mann et al., 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into this cell line (by calciumphosphate precipitation for example), the packaging sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is then collected, optionally concentrated,and used for gene transfer. Retroviral vectors are able to infect abroad variety of cell types. However, integration and/or stableexpression require the division of host cells (Paskind et al., 1975).

Concern with the use of defective retrovirus vectors is the potentialappearance of wild-type replication-competent virus in the packagingcells. This can result from recombination events in which the intactsequence from the recombinant virus inserts upstream from the gag, pol,env sequence integrated in the host cell genome. However, new packagingcell lines are now available that should greatly decrease the likelihoodof recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).

Gene delivery using second generation retroviral vectors has beenreported. Kasahara et al. (1994) prepared an engineered variant of theMoloney murine leukemia virus, that normally infects only mouse cells,and modified an envelope protein so that the virus specifically boundto, and infected, human cells bearing the erythropoietin (EPO) receptor.This was achieved by inserting a portion of the EPO sequence into anenvelope protein to create a chimeric protein with a new bindingspecificity.

4. Other Viral Vectors

Other viral vectors may be employed as expression constructs in thepresent invention. Vectors derived from viruses such as vaccinia virus(Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988),sindbis virus, cytomegalovirus and/or herpes simplex virus may beemployed. They offer several attractive features for various mammaliancells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986;Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, newinsight was gained into the structure-function relationship of differentviral sequences. In vitro studies showed that the virus could retain theability for helper-dependent packaging and reverse transcription despitethe deletion of up to 80% of its genome (Horwich et al., 1990). Thissuggested that large portions of the genome could be replaced withforeign genetic material. Chang et al. recently introduced thechloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virusgenome in the place of the polymerase, surface, and/or pre-surfacecoding sequences. It was cotransfected with wild-type virus into anavian hepatoma cell line. Culture media containing high titers of therecombinant virus were used to infect primary duckling hepatocytes.Stable CAT gene expression was detected for at least 24 days aftertransfection (Chang et al., 1991).

In certain further embodiments, the gene therapy vector will be HSV. Afactor that makes HSV an attractive vector is the size and organizationof the genome. Because HSV is large, incorporation of multiple genes andexpression cassettes is less problematic than in other smaller viralsystems. In addition, the availability of different viral controlsequences with varying performance (temporal, strength, etc.) makes itpossible to control expression to a greater extent than in othersystems. It also is an advantage that the virus has relatively fewspliced messages, further easing genetic manipulations. HSV also isrelatively easy to manipulate and/or can be grown to high titers. Thus,delivery is less of a problem, both in terms of volumes needed to attainsufficient MOI and in a lessened need for repeat dosings.

5. Modified Viruses

In still further embodiments of the present invention, the nucleic acidsto be delivered are housed within an infective virus that has beenengineered to express a specific binding ligand. The virus particle willthus bind specifically to the cognate receptors of the target cell anddeliver the contents to the cell. A novel approach designed to allowspecific targeting of retrovirus vectors was recently developed based onthe chemical modification of a retrovirus by the chemical addition oflactose residues to the viral envelope. This modification can permit thespecific infection of hepatocytes via sialoglycoprotein receptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand/or against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,1989). Using antibodies against major histocompatibility complex class Iand class II antigens, they demonstrated the infection of a variety ofhuman cells that bore those surface antigens with an ecotropic virus invitro (Roux et al., 1989).

B. Other Methods of DNA Delivery

In various embodiments of the invention, DNA is delivered to a cell asan expression construct. In order to effect expression of a geneconstruct, the expression construct must be delivered into a cell. Asdescribed herein, the preferred mechanism for delivery is via viralinfection, where the expression construct is encapsidated in aninfectious viral particle. However, several non-viral methods for thetransfer of expression constructs into cells also are contemplated bythe present invention. In one embodiment of the present invention, theexpression construct may consist only of naked recombinant DNA and/orplasmids. Transfer of the construct may be performed by any of themethods mentioned which physically and/or chemically permeabilize thecell membrane. Some of these techniques may be successfully adapted forin vivo and/or ex vivo use, as discussed below.

C. Liposome-Mediated Transfection

In a further embodiment of the invention, the expression construct maybe entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and/or an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and/orentrap water and/or dissolved solutes between the lipid bilayers (Ghoshand Bachhawat, 1991). Also contemplated is an expression constructcomplexed with Lipofectamine (Gibco BRL).

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). Wong et al. (1980) demonstrated thefeasibility of liposome-mediated delivery and/or expression of foreignDNA in cultured chick embryo, HeLa and hepatoma cells.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and/or promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed and/or employed in conjunction withnuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). Inyet further embodiments, the liposome may be complexed and/or employedin conjunction with both HVJ and HMG-1. In other embodiments, thedelivery vehicle may comprise a ligand and a liposome. Where a bacterialpromoter is employed in the DNA construct, it also will be desirable toinclude within the liposome an appropriate bacterial polymerase.

The inventors contemplate that neu-suppressing gene products can beintroduced into cells using liposome-mediated gene transfer. It isproposed that such constructs can be coupled with liposomes and directlyintroduced via a catheter, as described by Nabel et al. (1990). Byemploying these methods, the neu-suppressing gene products can beexpressed efficiently at a specific site in vivo, not just the liver andspleen cells which are accessible via intravenous injection. Therefore,this invention also encompasses compositions of DNA constructs encodinga neu-suppressing gene product formulated as a DNA/liposome complex andmethods of using such constructs.

As described in U.S. Pat. No. 5,641,484, liposomes are particularly wellsuited for the treatment of HER2/neu-mediated cancer.

Catatonic liposomes that are efficient transfection reagents for Bik foranimal cells can be prepared using the method of Gao et al. (1991). Gaoet al. describes a novel catatonic cholesterol derivative that can besynthesized in a single step. Liposomes made of this lipid arereportedly more efficient in transfection and less toxic to treatedcells than those made with the reagent Lipofectin. These lipids are amixture of DC-Chol (“3□(N-(N′N′-dimethylaminoethane)-carbamoylcholesterol”) and DOPE (“dioleoylphosphatidylethanolamine”). The stepsin producing these liposomes are as follows.

DC-Chol is synthesized by a simple reaction from cholesterylchloroformate and N,N-Dimethylethylenediamine. A solution of cholesterylchloroformate (2.25 g, 5 mmol in 5 ml dry chloroform) is added dropwiseto a solution of excess N,N-Dimethylethylenediamine (2 ml, 18.2 mmol in3 ml dry chloroform) at 0° C. Following removal of the solvent byevaporation, the residue is purified by recrystallization in absoluteethanol at 4° C. and dried in vacuo. The yield is a white powder ofDC-Chol.

Cationic liposomes are prepared by mixing 1.2 μmol of DC-Chol and 8.0μmol of DOPE in chloroform. This mixture is then dried, vacuumdesiccated, and resuspended in 1 ml sterol 20 mM Hepes buffer (pH 7.8)in a tube. After 24 hours of hydration at 4° C., the dispersion issonicated for 5-10 minutes in a sonicator form liposomes with an averagediameter of 150-200 nm.

To prepare a liposome/DNA complex, the inventors use the followingsteps. The DNA to be transfected is placed in DMEM/F12 medium in a ratioof 15 μg DNA to 50 μl DMEM/F12. DMEM/F12 is then used to dilute theDC-Chol/DOPE liposome mixture to a ratio of 50 μl DMEZM/F12 to 100 μlliposome. The DNA dilution and the liposome dilution are then gentlymixed, and incubated at 37° C. for 10 minutes. Following incubation, theDNA/liposome complex is ready for injection.

Liposomal transfection can be via liposomes composed of, for example,phosphatidylcholine (PC), phosphatidylserine (PS), cholesterol (Chol),N-[1-(2,3-dioleyloxy)propyl]-N,N-trimethylammonium chloride (DOTMA),dioleoylphosphatidylethanolamine (DOPE), and/or3.beta.[N—(N′N′-dimethylaminoethane)-carbarmoyl cholesterol (DC-Chol),as well as other lipids known to those of skill in the art. Those ofskill in the art will recognize that there are a variety of liposomaltransfection techniques that will be useful in the present invention.Among these techniques are those described in Nicolau et al., 1987,Nabel et al., 1990, and Gao et al., 1991. In a specific embodiment, theliposomes comprise DC-Chol. More particularly, the inventors theliposomes comprise DC-Chol and DOPE that have been prepared followingthe teaching of Gao et al. (1991) in the manner described in thePreferred Embodiments Section. The inventors also anticipate utility forliposomes comprised of DOTMA, such as those that are availablecommercially under the trademark LipofectinTM, from Vical, Inc., in SanDiego, Calif.

Liposomes may be introduced into contact with cells to be transfected bya variety of methods. In cell culture, the liposome-DNA complex cansimply be dispersed in the cell culture solution. For application invivo, liposome-DNA complex are typically injected. Intravenous injectionallow liposome-mediated transfer of DNA complex, for example, the liverand the spleen. In order to allow transfection of DNA into cells thatare not accessible through intravenous injection, it is possible todirectly inject the liposome-DNA complexes into a specific location inan animal's body. For example, Nabel et al. teach injection via acatheter into the arterial wall. In another example, the inventors haveused intraperitoneal injection to allow for gene transfer into mice.

The present invention also contemplates compositions comprising aliposomal complex. This liposomal complex will comprise a lipidcomponent and a DNA segment encoding a nucleic acid encoding a mutantform of Bik. The nucleic acid encoding the mutant form of Bik employedin the liposomal complex can be, for example, one that encodes Bik-T145Aor Bik-T145D.

The lipid employed to make the liposomal complex can be any of theabove-discussed lipids. In particular, DOTMA, DOPE, and/or DC-Chol mayform all or part of the liposomal complex. The inventors have hadparticular success with complexes comprising DC-Chol. In a preferredembodiment, the lipid will comprise DC-Chol and DOPE. While any ratio ofDC-Chol to DOPE is anticipated to have utility, it is anticipated thatthose comprising a ratio of DC-Chol:DOPE between 1:20 and 20:1 will beparticularly advantageous. The inventors have found that liposomesprepared from a ratio of DC-Chol:DOPE of about 1:10 to about 1:5 havebeen useful.

In a specific embodiment, one employs the smallest region needed toenhance retention of Bik in the nucleus of a cell so that one is notintroducing unnecessary DNA into cells which receive a Bik geneconstruct. Techniques well known to those of skill in the art, such asthe use of restriction enzymes, will allow for the generation of smallregions of Bik. The ability of these regions to inhibit neu can easilybe determined by the assays reported in the Examples.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinatin virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention. Where a bacterial promoter is employed in the DNA construct,it also will be desirable to include within the liposome an appropriatebacterial polymerase.

D. Electroporation

In certain embodiments of the present invention, the expressionconstruct is introduced into the cell via electroporation.Electroporation involves the exposure of a suspension of cells and/orDNA to a high-voltage electric discharge.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected withhumankappa-immunoglobulin genes (Potter et al., 1984), and/or rathepatocytes have been transfected with the chloramphenicolacetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.

E. Calcium Phosphate and/or DEAE-Dextran

In other embodiments of the present invention, the expression constructis introduced to the cells using calcium phosphate precipitation.HumanKB cells have been transfected with adenovirus 5 DNA (Graham andVan Der Eb, 1973) using this technique. Also in this manner, mouseL(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and/or HeLa cells weretransfected with a neomycin marker gene (Chen and Okayama, 1987), and/orrat hepatocytes were transfected with a variety of marker genes (Rippeet al., 1990).

In another embodiment, the expression construct is delivered into thecell using DEAE-dextran followed by polyethylene glycol. In this manner,reporter plasmids were introduced into mouse myeloma and/orerythroleukemia cells (Gopal, 1985).

F. Particle Bombardment

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and/or entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungstenand/or gold beads.

G. Direct Microinjection and/or Sonication Loading

Further embodiments of the present invention include the introduction ofthe expression construct by direct microinjection and/or sonicationloading. Direct microinjection has been used to introduce nucleic acidconstructs into Xenopus oocytes (Harland and Weintraub, 1985), and/orLTK-fibroblasts have been transfected with the thymidine kinase gene bysonication loading (Fechheimer et al., 1987).

H. Adenoviral Assisted Transfection

In certain embodiments of the present invention, the expressionconstruct is introduced into the cell using adenovirus assistedtransfection. Increased transfection efficiencies have been reported incell systems using adenovirus coupled systems (Kelleher and Vos, 1994;Cotten et al., 1992; Curiel, 1994).

V. Combination Treatments

In order to increase the effectiveness of a therapeutic gene productencoded by a construct comprising a promoter of the invention, it may bedesirable to combine these compositions with other agents effective inthe treatment of hyperproliferative disease, such as anti-cancer agents.An “anti-cancer” agent is capable of negatively affecting cancer in asubject, for example, by killing cancer cells, inducing apoptosis incancer cells, reducing the growth rate of cancer cells, reducing theincidence or number of metastases, reducing tumor size, inhibiting tumorgrowth, reducing the blood supply to a tumor or cancer cells, promotingan immune response against cancer cells or a tumor, preventing orinhibiting the progression of cancer, or increasing the lifespan of asubject with cancer. More generally, these other compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theexpression construct and the agent(s) or multiple factor(s) at the sametime. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes the expressionconstruct and the other includes the second agent(s).

Therapy with the methods and compositions of the present invention canbe used in conjunction with chemotherapeutic, radiotherapeutic,immunotherapeutic therapy, surgery, hormonal therapy, or additional genetherapy with other pro-apoptotic or cell cycle regulating agents. Genetherapy with the inventive promoters and/or gene therapy in addition tothe inventive compositions and methods may utilize inducers of cellularproliferation; antisense sequences for inducers of cellularproliferation; inhibitors of cellular proliferation, such as p53, p16,Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1, p73, VHL,MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16 fusions, Bik/p27 fusions,anti-thrombotic genes (e.g., COX-1, TFPI), PGS, Dp, E2F, ras, myc, neu,raf, erb, fins, trk, ret, gsp, hst, abl, E1A, p300, genes involved inangiogenesis (e.g., VEGF, FGF, thrombospondin, BAI-1, GDAIF, or theirreceptors) or MCC; and/or regulators of programmed cell death, such asthose that counteract Bcl-2 function and promote cell death (e.g., Bax,Bak, Bik, Bim, Bid, Bad, Harakiri).

VI. Pharmaceutical Preparations

Pharmaceutical compositions of the present invention comprise aneffective amount of a construct comprising control sequences of thepresent invention that regulate expression of a therapeutic gene productand, in specific embodiment one or more additional agents, dissolved ordispersed in a pharmaceutically acceptable carrier or excipient. Thephrases “pharmaceutical or pharmacologically acceptable” refers tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, suchas, for example, a human, as appropriate. The preparation of anpharmaceutical composition that contains at least one constructcomprising the inventive control sequences that regulate expression of atherapeutic polynucleotide and, in some embodiments one or moreadditional active ingredients, will be known to those of skill in theart in light of the present disclosure, as exemplified by Remington'sPharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,incorporated herein by reference. Moreover, for animal (e.g., human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, binders, excipients, disintegration agents, lubricants,sweetening agents, flavoring agents, dyes, such like materials andcombinations thereof, as would be known to one of ordinary skill in theart (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.Mack Printing Company, 1990, pp. 1289-1329, incorporated herein byreference). Except insofar as any conventional carrier is incompatiblewith the active ingredient, its use in the therapeutic or pharmaceuticalcompositions is contemplated. In a specific embodiment, the mutant Bikcomposition is administered in a liposome.

The therapeutic construct comprising the tissue-specific controlsequences may comprise different types of carriers depending on whetherit is to be administered in solid, liquid or aerosol form, and whetherit need to be sterile for such routes of administration as injection.The present invention can be administered intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, rectally, topically,intratumorally, intramuscularly, intraperitoneally, subcutaneously,intravesicularlly, mucosally, intrapericardially, orally, topically,locally, using aerosol, injection, infusion, continuous infusion,localized perfusion bathing target cells directly, via a catheter, via alavage, in cremes, in lipid compositions (e.g., liposomes), or by othermethod or any combination of the forgoing as would be known to one ofordinary skill in the art (see, for example, Remington's PharmaceuticalSciences, 18th Ed. Mack Printing Company, 1990, incorporated herein byreference).

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. The practitioner responsible for administration will, inany event, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. In other non-limitingexamples, a dose may also comprise from about 1 microgram/kg/bodyweight, about 5 microgram/kg/body weight, about 10 microgram/kg/bodyweight, about 50 microgram/kg/body weight, about 100 microgram/kg/bodyweight, about 200 microgram/kg/body weight, about 350 microgram/kg/bodyweight, about 500 microgram/kg/body weight, about 1 milligram/kg/bodyweight, about 5 milligram/kg/body weight, about 10 milligram/kg/bodyweight, about 50 milligram/kg/body weight, about 100 milligram/kg/bodyweight, about 200 milligram/kg/body weight, about 350 milligram/kg/bodyweight, about 500 milligram/kg/body weight, to about 1000 mg/kg/bodyweight or more per administration, and any range derivable therein. Innon-limiting examples of a derivable range from the numbers listedherein, a range of about 5 mg/kg/body weight to about 100 mg/kg/bodyweight, about 5 microgram/kg/body weight to about 500 milligram/kg/bodyweight, etc., can be administered, based on the numbers described above.

In any case, the composition may comprise various antioxidants to retardoxidation of one or more component. Additionally, the prevention of theaction of microorganisms can be brought about by preservatives such asvarious antibacterial and antifungal agents, including but not limitedto parabens (e.g., methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, thimerosal or combinations thereof.

The therapeutic construct may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine.

In embodiments where the composition is in a liquid form, a carrier canbe a solvent or dispersion medium comprising but not limited to, water,ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethyleneglycol, etc), lipids (e.g., triglycerides, vegetable oils, liposomes)and combinations thereof. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin; by the maintenanceof the required particle size by dispersion in carriers such as, forexample liquid polyol or lipids; by the use of surfactants such as, forexample hydroxypropylcellulose; or combinations thereof such methods. Inmany cases, it will be preferable to include isotonic agents, such as,for example, sugars, sodium chloride or combinations thereof.

In other embodiments, one may use eye drops, nasal solutions or sprays,aerosols, mouthwashes, or inhalants in the present invention. Suchcompositions are generally designed to be compatible with the targettissue type. In a non-limiting example, nasal solutions are usuallyaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions are prepared so that they are similarin many respects to nasal secretions, so that normal ciliary action ismaintained. Thus, in preferred embodiments the aqueous nasal solutionsusually are isotonic or slightly buffered to maintain a pH of about 5.5to about 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations, drugs, or appropriate drug stabilizers,if required, may be included in the formulation. For example, variouscommercial nasal preparations are known and include drugs such asantibiotics or antihistamines.

In certain embodiments the Bik mutant form is prepared foradministration by such routes as oral ingestion. In these embodiments,the solid composition may comprise, for example, solutions, suspensions,emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatincapsules), sustained release formulations, buccal compositions, troches,elixirs, suspensions, syrups, wafers, mouthwashes, or combinationsthereof. Oral compositions may be incorporated directly with the food ofthe diet. Preferred carriers for oral administration comprise inertdiluents, assimilable edible carriers or combinations thereof. In otheraspects of the invention, the oral composition may be prepared as asyrup or elixir. A syrup or elixir, and may comprise, for example, atleast one active agent, a sweetening agent, a preservative, a flavoringagent, a dye, a preservative, or combinations thereof.

In certain preferred embodiments an oral composition may comprise one ormore binders, excipients, disintegration agents, lubricants, flavoringagents, and combinations thereof. In certain embodiments, a compositionmay comprise one or more of the following: a binder, such as, forexample, gum tragacanth, acacia, cornstarch, gelatin or combinationsthereof; an excipient, such as, for example, dicalcium phosphate,mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate or combinations thereof; a disintegratingagent, such as, for example, corn starch, potato starch, alginic acid orcombinations thereof; a lubricant, such as, for example, magnesiumstearate; a sweetening agent, such as, for example, sucrose, lactose,saccharin or combinations thereof; a flavoring agent, such as, forexample peppermint, oil of wintergreen, cherry flavoring, orangeflavoring, etc.; or combinations thereof the foregoing. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, carriers such as a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar or both.

Additional formulations which are suitable for other modes ofadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum, vagina or urethra. After insertion, suppositoriessoften, melt or dissolve in the cavity fluids. In general, forsuppositories, traditional carriers may include, for example,polyalkylene glycols, triglycerides or combinations thereof. In certainembodiments, suppositories may be formed from mixtures containing, forexample, the active ingredient in the range of about 0.5% to about 10%,and preferably about 1% to about 2%.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle that contains the basic dispersion medium and/or the otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, suspensions or emulsion, the preferredmethods of preparation are vacuum-drying or freeze-drying techniqueswhich yield a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessary andthe liquid diluent first rendered isotonic prior to injection withsufficient saline or glucose. The preparation of highly concentratedcompositions for direct injection is also contemplated, where the use ofDMSO as solvent is envisioned to result in extremely rapid penetration,delivering high concentrations of the active agents to a small area.

The composition must be stable under the conditions of manufacture andstorage, and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi. It will be appreciated thatendotoxin contamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein.

In particular embodiments, prolonged absorption of an injectablecomposition can be brought about by the use in the compositions ofagents delaying absorption, such as, for example, aluminum monostearate,gelatin or combinations thereof.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Breast Cancer Tissue-Specific Expression

Current breast cancer (BC) therapies, such as chemotherapy (CT) andradiotherapy have low selectivity for tumor cells and side effects fornormal tissues. To minimize the side effects, these therapies aregenerally given in an intermittent manner, allowing normal cells torecover between treatment cycles. However, during the recovery period,some surviving cancer cells become more resistant to the treatmentbecause of gene mutation. Consequently, cancer recurrence or progressionmay occur. Tumor-targeting gene therapy can minimize treatment sideeffects and the risk of developing resistance by acting on thetumor-specific signaling pathways. In the present invention, breastcancer-specific promoters are used for breast cancer-targeting genetherapy of the exemplary therapeutic polynucleotide, mutant Bik.

The tumorigenesis and progression of breast cancer involve a series ofgenetic changes. The specifically activated genes in tumors are goodtargets of therapies. As the first step, the present inventors collectedand reviewed published data from cDNA microarray and SAGE, andidentified six genes specifically upregulated in breast cancer cells asshown in Table 1.

TABLE 1 Genes upregulated specifically in breast cancer cells. Gene T/NPCI (Max = −0.4) Transferrin receptor >20 −0.3 B-Myb >20 NDCeruloplasmin >10 −0.35 X-box Binding protein 1  >8 ND γ-glutamylhydrolase conjugase >20 −0.33 Topoisomerase IIα >20 ND

T/N, gene expression ratio of tumor to normal cells. PCI, prognosticcorrelation index. The negative value of PCI indicates worse prognosis.The maximum value of negative PCI is −0.4.

Among them, the promoters of transferrin receptor (TR), B-Myb,ceruloplasmin, and topoisomerase IIα (topoIIα) were subcloned intoluciferase reporter vectors and tested using reporter assays with normaland cancer cell lines. TopoIIα and TR promoters have the highestactivity in breast cancer cells relative to normal breast epithelial184A1 cells. Therefore, the present inventors further pursued breastcancer-specific cis-elements in these two promoters.

TopoIIα catalyzes topological changes of DNA to release its tensiongenerated during replication, transcription, and chromosome segregation.It is also the target of several anticancer agents, such asanthracyclines (e.g. etoposide and doxorubicin). Many studies have shownthat topoIIα level correlates with the sensitivity of cancer cells toanthracyclines. In addition, topoIIα is a poor prognostic marker forbreast cancer, brain tumors, hepatoma, etc. The regulation of topoIIαexpression is strictly cell cycle-dependent: its mRNA level and promoteractivity are very low at G0/G1 phase, begin to rise in late S phase, andreach peak at G2/M phase. TopoIIα is a TATA-less promoter containingfive Inverted CCAAT Boxes (ICB) and two GC boxes. It is regulated byheat shock, cell cycle stages and p53. NF-Y (also known as CBF) binds toICBs in the promoter and is required for topoIIα transcription duringcell cycle. Taken together, this indicates that the topoIIα promoterresponds to oncogenic signaling, in some embodiments, and ICB sites andtheir binding factors could play a very important role in itsBC-specific activity.

In one embodiment of the present invention, transferrin receptor isutilized in composite promoters for the present invention. TR is amembrane receptor that interacts with iron-bound transferrin,facilitating the transport of iron across the cell membrane. A higher TRmRNA level correlates with poor differentiation, greater invasiveness,and high proliferative index of breast cancer cells. In vitro studieshave demonstrated that antisense oligonucleotide targeting TR mRNA canspecifically inhibit the growth of human breast cancer cells withoutaffecting normal breast cells, indicating TR plays a crucial role inbreast cancer development. Even though regulation in mRNA stability isthe primary determinant of TR expression in normal cells, many studiesrevealed that TR transcription is highly activated in proliferatingcells and by several oncogenic signals. The core region of TR promotercontains a TATA box, GC box, AP-1/CRE site, and HRE, which responds tohypoxia. Signals like proliferation, hypoxia, iron shortage, anddifferentiation can activate this segment of TR promoter.

As shown below, the present inventors utilized these exemplary breastcancer-specific CT90 and CTR116 promoters in conjunction with apolynucleotide encoding a mutant Bik polypeptide, in particularembodiments comprised in liposomes. The exemplary CT90-driving BikDDtherapeutic vector (CT90-BikDD) selectively killed BC cells in vitro andsuppressed the growth of breast tumor xenograft in a mouse model. Takentogether, these results indicated that CT90 and CTR116 are effective,strong breast cancer-specific promoters that are useful to controlmutant Bik gene expression in breast cancer-targeting gene therapy.

Identification of Core BC-Specific Segments in topoIIα Promoter

To identify one or more segments comprising the cis-elements requiredfor its breast cancer-specificity, the present inventors generated aseries of topoIIα promoter deletion mutants by, for example, polymerasechain reaction (PCR). Each mutant contained different number of ICBsite(s) ranging from one to five and was subcloned into luciferaseexpression vector. These reporter constructs were then transientlytransfected in various cell lines, and their promoter activities wereexamined using a luciferase reporter assay. A 580-bp (referred to asfull-length) promoter had the highest activity among all the deletionmutants and was activated mostly in breast cancer cells (FIG. 1).However, the shortest promoter segment, which spanned 90 base pairs fromthe transcription start site and comprised the first ICB site, possessedminimal promoter activity and still retained breast cancer specificity(FIG. 2). This result indicated that the 90-bp segment comprised corebreast cancer-specific elements.

Enhancement of topoIIα Promoter Activity with CMV Enhancer Sequence

The activities of all the topoIIα promoter deletion mutants were as lowas less than 10% of CMV promoter activity. To enhance the promoteractivity for clinical application, the present inventors obtained theexemplary CMV enhancer sequence from the pCDNA3.1 plasmid by PCR,connected it directly upstream of the full-length and 90-bp topoIIαpromoter deletion mutants, and then subcloned it into luciferasereporter vectors. Two exemplary composite promoters were referred to asCT572 and CT90, respectively. Compared to the original promoter deletionmutants, the activity of CT572 and CT90 was dramatically elevated andcomparable to the CMV promoter, but still retained their breast cancerspecificity (FIG. 3). CT90 promoter has a much higher activity thanCT572, and it is nearly strong as the CMV promoter in some cell lines(FIG. 3). Therefore, CT90 was utilized for the in vivo test. Breastcancer MDA-MB-231 cells were inoculated into mammary fat pad of nudemice. Four weeks after inoculation, each mouse in the experiment groupreceived 50 μg of liposome-complexed CT90-luciferase vector delivered byintravenous injection from tail vein, and CMV-luciferase vector wasgiven to mice in the control group. Forty-eight hours after injection,the mice were sacrificed, and the promoter activity was examined by aluciferase assay in tumor, heart, lung, liver, spleen, kidney, andmuscle (FIG. 4). CT90 had higher activity than CMV promoter in tumor.However, in normal tissues, the activity of CT90 was much lower than theCMV promoter. This indicated that the CT90 promoter has BC-specificactivity in vivo.

Anti-Tumor Effect of CT90-BikDD

To characterize CT90 in breast cancer-targeting gene therapy, atherapeutic construct in which CT90 drives BikDD expression wasgenerated and is hereinafter referred to as CT90-BikDD. This constructwas co-transfected with a luciferase reporter vector into breast cancercell lines MDA-MB-231 and 468, and the normal breast epithelium cellline 184A1, and then the cell-killing effect was determined by aluciferase vitality assay. The CMV promoter-drivin BikDD vector(CMV-BikDD) and empty vector were used as positive and negativecontrols, respectively. While CMV-BikDD killed all three cell lines to anearly equal extent, CT90-BikDD killed breast cancer cellspreferentially (FIG. 5), indicating that the killing effect ofCT90-BikDD is selective for breast cancer cells. Therefore, CT90 isuseful in the breast cancer-targeting gene therapy.

Next, the anti-tumor effect of this breast cancer-targeting gene therapywas characterized in vivo. One week after inoculating breast cancerMDA-MB-231 cells into mammary fat pads, the nude mice were treated onceper week with liposome-complexed CT90-BikDD (therapeutic group),CMV-BikDD (positive control), and CMV-PGL3 (mock treatment), or dextrosebuffer D5W as a no-treatment control. Each mouse was intravenouslyinjected with 15 μg of liposome-complexed DNA construct, once per week,and tumor size was measured regularly. The CT90-BikDD group showed asuperior tumor suppressive effect compared to CMV-BikDD or CMV-PGL3(FIG. 6).

Identification of Core Breast Cancer-Specific Segments in TransferrinReceptor Promoter

To identify the segment containing the cis-elements required for itsbreast cancer-specificity, a series of TR promoter deletion mutants(1412-, 1123-, and 193-bp upstream to the transcription starting site)were generated by PCR and subcloned into a luciferase expression vector.These reporter constructs were then transiently transfected in variouscell lines as mentioned above, and their promoter activities wereexamined using a luciferase reporter assay. The 193-bp segment (187 bpupstream to the transcriptional starting site) had the highest activityand breast cancer specificity in vitro (FIG. 7).

Enhancement of TR Core Promoter Activity with CMV Enhancer Sequence

To narrow down the range to a core breast cancer-specific promoter, the5′-end of the 193-bp segment of the TR promoter was further deleted116-bp upstream of the transcription start site and connected to a CMVenhancer sequence, as described above for CT90. This composite promoter,herein referred to as CTR116, was subcloned into a luciferase reportervector and transfected into the cell lines. After transfection, thecells were treated with normoxic or hypoxic conditions (94% N₂, 5% CO₂,1% O₂ for 20 hours), and the promoter activity of CTR116 was determinedby a luciferase assay. Compared to the original deletion mutantpromoter, the activity of CTR116 was clearly elevated while retainingits breast cancer specificity. Moreover, its activity can be furtherinduced by hypoxic treatment to become comparable to CMV promoter (FIG.8). This is the first demonstration that at least part of the TRpromoter possesses breast cancer specificity, and a CMV promoterenhancer can enhance its activity without interfering with hypoxiainduction.

In further embodiments of the present invention, the respective CT90 andCTR116 elements are narrowed further to identify even smaller segmentswithin that retain breast cancer-specific expression activity. Forexample, deletion constructs may be made of these respective regions,and their tissue specificity is tested to identify the smaller segmentsthat maintain the ability to direct expression in breast cancer tissue.

The CT90 Promoter Drives Expression of BikDD in Comparable Level to thatfrom the CMV Promoter

To further characterize the activity of the CT90 promoter for inducinggene expression at a similar level as the CMV promoter, which possessesstrong activity and is widely used in systemic gene therapy, CT90-BikDDor CMV-BikDD construct were transfected into MCF-7 breast cancer cellsby electroporation. 24 hours after transfection, the cells wereharvested and lysed for Western blot. As shown in FIG. 14, theexpression level of BikDD protein from the CT90 promoter is slightlyhigher than that from the CMV promoter. This result indicates the CT90promoter possesses strong activity in breast cancer cells.

CT90-BikDD Lipoplex Selectively Suppressed Breast Tumor Growth andProlonged Survival in an Orthotopic Mouse Model

To characterize whether systemically delivered liposome-complexedCT90-BikDD (CT90-BikDD lipoplex) could direct selective BikDD expressionin vivo, MDA-MB-231 (FIG. 15A) or MDA-MB-468 cells (FIG. 15B) wereinoculated in the mammary fat pad of female nude mice to form breasttumors. The tumor bearing mice were then received treatments ofliposome-delivered CT90-BikDD, CMV-BikDD, pGL3 vector (mock treatment),or no treatment (D5W), with 8 mice in each treatment group. Lipoplex ofdifferent DNA constructs was intravenously injected to mice in thecorresponding treatment groups once per week. The tumor growth inCT90-BikDD and CMV-BikDD treatment groups was suppressed significantly(p<0.05 in T-test) compared with results in both mock treatment (pGL3)and no treatment (D5W) groups (FIGS. 15A and 15B). As shown in FIG. 15,the mean survival time of mice in CT90-BikDD, CMV-BikDD, pGL3, and D5Wgroups are 25.25±1.83, 23.25±1.74, 17.5±1.3 and 16.25±0.98 weeks,respectively. Both CT90-BikDD and CMV-BikDD treatments yield significantsurvival benefit for MDA-MB-231 xenograft-bearing mice compared to theno treatment (D5W) or mock treatment (pGL3) group, indicating thatCT90-BikDD provided comparable therapeutic effect as CMV-BikDD (FIG. 16,lower panel).

CT90-BikDD Possesses Anti-Tumor Activity and Minimum Side Effect inNormal Tissues

The present inventors then further characterize whether the in vivodifferential expression profile of CT90-luc and CMV-luc could bereflected by CT90-BikDD and CMV-BikDD. To address this issue, expressionof BikDD mRNA in tumor and heart was examined by in situ hybridization(FIG. 17). The deep brown staining all over the heart tissue from aCMV-BikDD-treated mouse indicated a very high expression level of BikDDfrom CMV promoter (FIG. 17A, upper left panel). On the contrary,CT90-BikDD treatment induced relatively weak BikDD expression in theheart tissues (FIG. 17A, upper right panel). No significant differencein the density and expression level could be detected between the tumorspecimens from CT90-BikDD and CMV-BikDD group (FIG. 17B). The negativecontrol using sense BikDD showed no brown staining in these experiments(FIGS. 17A and 17B, lower panels), indicating that the positive signalsin the antisense groups came from BikDD expression. These datademonstrate that systemically administrated CT90-BikDD lipoplex candirect the selective BikDD expression in breast tumor. Importantly,expression of BikDD in the normal organ such as heart is much lower inthe CT90-BikDD-treated mice than CMV-BikDD-treated mice. Thus, incomparison with CMV-BikDD, CT90-BikDD possesses comparable anti-tumoractivity and will have minimum side effects induced by its expression innormal tissues.

ICB1 Can Mediate Activation of topoIIα Promoter by Cdk2/cyclin ASpecifically in Breast Cancer Cells

To enhance the basal activity of topoIIα promoter in breast cancercells, in a specific embodiment a strong enhancer is linked to it.However, in certain aspects due to the possible interference from someof the regulatory elements in the topoIIα promoter, the addition of thestrong enhancer might not achieve high basal activity and could evenlose breast cancer specificity (see later in FIG. 26). In order todevelop a TSP possessing both specificity and high basal activity inbreast cancer cells, the present inventors reasoned that a minimalbreast cancer-specific element linking to an enhancer would have abetter successful opportunity. Thus, we set out to identify a breastcancer-specific element in the topoIIα promoter. To this end, we firstexplored the possible breast cancer-specific signal to activate topoIIαpromoter, then looked for the cis-element that mediated this signaling.TopoIIα expression is upregulated during S phase and peaks during G2/M(Woessner et al., 1991). Since the essential cell-cycle regulator Cdk2is activated during S and G2 phases (Vermeulen et al., 2003), thepresent inventors asked whether topoIIα promoter activity might beupregulated by Cdk2. To this end, the regulation of cdk2 on topoIIαpromoter activity was examined by cotransfecting a dominant-negativeCdk2 mutant (Cdk2-dn) (van den Heuvel & Harlow, 1993) and topoIIαpromoter reporter construct in a reporter assay. Cdk2-dn suppressedtopoIIα promoter activity in three different breast cancer cells but notin normal breast epithelial cells (184A1), lung cancer cells (A549), ornonmalignant hepatocytes (Chang liver) (Castagnetta et al., 2003) (FIG.25A). Since Cdk2 associates with cyclin E and cyclin A in late G1 and Sphase, respectively, we investigated which one could activate topoIIαpromoter. Expression of cyclin A significantly induced activation of thetopoIIα promoter in breast cancer cells but not in other cell types(FIG. 25B). On the contrary, cyclin E had minimum effect on the topoIIαpromoter in breast cancer cells (data not shown). These data suggestthat cyclin A/Cdk2 activates the topoIIα promoter in a breastcancer-specific manner.

Following the results above, the cyclin A/Cdk2-responding element in thetopoIIα promoter might be the potential breast cancer-specific element.To identify this element, we generated a series of deletion mutants ofthe topoIIα promoter, including −182, −90, −60, which contain three,one, and no ICB(s), respectively (FIG. 25C) (Adachi et al., 2000; Falcket al., 1999; Hochhauser et al., 1992). Their responses to cyclin Aactivation were examined in the breast cancer cell line SK-BR3 (FIG.25D, left panel). Cyclin A can activate the full-length promoter as wellas its −182 and −90 deletion mutants that harbor at least one ICB (−572,−182, and −90 in left panel of FIG. 2D, p<0.05), but not the −60deletion mutant which lacks any ICB (−60 in left panel of FIG. 2D).Similar results were obtained when these experiments were performed inother two breast cancer cell lines MDA-MB-468 and MDA-MB-231 (data notshown), indicating ICBs could be important for mediating the cyclin Asignal. This point was further supported by the fact that the mutationof ICB1 in full-length topoIIα promoter (FIG. 2C, lowest one) resultedin the greater reduction in the response to cyclin A (FIG. 2E, rightpanel), suggesting that ICB1 in the topoIIα promoter is required forfull response to cyclin A/cdk2 activation and may represent a minimalbreast cancer-specific element.

To validate the role of ICB1 in mediating breast cancer specificity oftopoIIα promoter, the activity of the −90 deletion mutant was examinedin a panel of cell lines since it contains only one ICB site. Similar tothe full-length promoter, the −90 promoter activity was higher in mostbreast cancer cell lines than in other types of cells (FIG. 25E, upperpanel), indicating it remains the breast cancer specificity. However,the activity ratio of the −90 promoter to the CMV promoter (−90/CMV)again showed its low basal activity in breast cancer cells (<0.5% inlower panel of FIG. 25E). Since most other regulatory elements have beendeleted in the −90 promoter, it may be easier to be engineered intohigher basal activity in breast cancer and remains the specificity.

CT90 Promoter, −90 topoIIα Promoter Potentiated with Enhancer Sequenceof CMV Promoter, Possesses a Stronger Specific Activity in Breast CancerCells In Vitro and In Vivo

To enhance basal activity in breast cancer cells, we connected theenhancer sequence of the CMV promoter (CMV enhancer) (Xu et al., 2001)to three ICB-harboring promoters: full-length topoIIα promoter and its−182 and −90 deletion mutants. The composite promoters were designatedas CT572, CT182, and CT90, respectively (FIG. 26A). Interestingly, amongthese composite promoters, CT90 has highest activity in breast cancercells relative to the normal 184A1 cells (FIG. 26B, the reporteractivity is shown by log scale). Moreover, the activity of CT90 iscomparable to the CMV promoter in breast cancer cells (FIG. 26C). CT572and CT182 have lower specificity for breast cancer cells than CT90 (FIG.26B), and their activities were enhanced in a very limited extent (FIG.26C), suggesting that some elements other than ICB1 in topoIIα promotermay interfere the CMV enhancer activity.

To test whether CT90 possesses sufficient specificity and activity forsystemic liposome gene therapy, we further examine its activity in vivo.The luciferase constructs driven by the CT90 or CMV promoter (CT90-lucand CMV-luc, respectively) were complexed with DOTAP:Chol liposome, thenintravenously injected into mice carrying either MDA-MB-231 breastcancer xenograft. The promoter activity in the normal and tumor tissueswas determined by reporter assay (upper panels of FIG. 26D). Asmentioned earlier, when cationic liposome-DNA complex is administratedthrough i.v. injection into animal, lung and heart will uptake anessential portion of liposome, resulting in the higher gene expressionlevel in these two organs (Barron, 1999; Li et al., 1998; Lu et al.,2002; Templeton et al., 1997). Likewise, the biodistribution of reporteractivities from CMV-luc showed similar pattern: the highest in lung,second and third highest in heart and liver, lower in all other tissues(FIG. 26D, upper panels). This indicates that the reporter activity fromCMV-luc represents the gene delivery efficiency of liposome in ouranimal models. To evaluate the specificity of CT90 in vivo, the liposomeuptake efficiency was normalized in different tissues (Barron, 1999; Liet al., 1998; Lu et al., 2002; Templeton et al., 1997) by calculatingthe activity ratio of CT90 to the CMV promoter (CT90/CMV in the lowerpanels of FIG. 26D). The normalized CT90 activity was higher in thetumor, but much lower in the normal organs, especially in the majorliposome-trapping organs heart, lung, and liver (FIG. 26D). Similarbiodistribution of CMV-luc and CT90-luc activity were observed inanother mouse model carrying MDA-MB-468 breast cancer xenograft (datanot shown). When another liposome SN (Li et al., 2003; Zou et al., 2002)was used to deliver DNA construct, CT90 still showed similar tumorspecificity (data not shown). It needs to be mentioned here that theliposome delivery efficiency to spleen, kidney, and muscle is much lowerthan that to lung, heart, and liver. Therefore, in these experiments,the expression of delivered gene in the latter three organs is minimumand likely negligible in the comparison of promoter activity. To ruleout the influence from gene delivery efficiency, the CMV-luc or CT90-lucis delivered by DOTAP:Chol liposome into MDA-MB-231 tumor-bearing micethrough intratumoral injection. The luciferase activities from the CT90promoter are still higher in tumor, and lower in lung or heart, thanthose from the CMV promoter (FIG. 26E), indicating CT90 is atumor-specific promoter in vivo. Interestingly, the breast cancerspecificity and activity of CT90 in vivo are higher than those in vitro(FIGS. 26C and 26D). Such phenomenon that in vivo liposome deliveryincreases the promoter specificity compared to in vitro delivery hasalso been observed in other study (Lu et al., 2002). These resultsdemonstrated that CT90 is a stronger breast cancer-specific promoterthat is suitable for breast cancer targeting in the gene therapysetting.

Example 2 Pancreatic Cancer-Specific Expression

The present inventors utilized pancreatic cancer-specific promotersequences to control expression of a polynucleotide encoding a mutantBik polypeptide. Exemplary methods and compositions directed to thisgoal are described in this Example.

Cell Lines

Human pancreatic cancer (PANC-1, CAPAN-1 and AsPC-1), prostate cancer(LNCaP and PC-3) cell lines, immortalized normal lung fibroblast (WI-38)and mammary epithelial (184A1) cells were obtained from the AmericanType Culture Collection (ATCC, Rockville, Md.). Immortalized humanpancreatic cells E6E7 was kindly provided by Dr. Paul Chiao (Universityof Texas M. D. Anderson Cancer Center, Houston, Tex.). Chang liver cellsand ovarian cancer cell line SKOV3.ip1 were also used. PANC-1, CAPAN-1,AsPC-1, WI-38, and SKOV3.ip1, and Chang liver cells were cultured withDMEM/F12 medium supplemented with 10% fetal bovine serum (FBS),penicillin (100 units/ml) and streptomycin (100 mg/ml) (Invitrogen,Carlsbad, Calif.). LNCaP and PC-3 cells were maintained in RPMI 1640medium (Invitrogen) with 10% (FBS), penicillin and streptomycin. 184A1cells were maintained in complete mammary epithelium growth medium(MEGM) (Cambrex, Walkerswille, Md.) and E6E7 cells in Keratiocyte-SFMsupplemented with epidermal growth factor, pituitary extract(Invitrogen).

Constructs

The CCKAR promoters (−726 to +1; primers CCKAR-p1 and CCKAR-p2), uPARpromoter (−812 to +1; primers uPAR-p1, and uPAR-p2), RDC-1 promoter(−881 to +1; primers RDC-p1, and RDC-p2), CTRB1 promoter (−946 to +1;primers CTRB1-p1, and CTRB1-p2) and CPA1 promoter (−1010 to +108;CPA1-p1 and CPA1-p2) were obtained by PCR amplification of PANC-1genomic DNA using primers shown in Table 2.

TABLE 2 The sequence of potential pancreatic cancer specific promotersCCKAR- 5′-AGGACCCAGGTACCTATGTTCAAAAGTGCCTC-3′ p1: (SEQ ID NO: 1) CCKAR-5′-CCTTGCCTGCTGCTTTCCACCAAGTGCT-3′ p2: (SEQ ID NO: 2) RDC1-p1:5′-CAGGTTGGGAAAATGGTCAGCCCTCCTGAAA-3′ (SEQ ID NO: 3) RDC-p2:5′-CGTTCTGAGGCGGGCAATCAAATGACCTAT-3′ (SEQ ID NO: 4) uPAR-p15′-CCCGCTAGCCTAATTTTATTTTATTTTTAATTC-3′ (SEQ ID NO: 5) uPAR-p25′-CCCCTCGAGGTATTTTGGAAAAATGTCCTTATCTAG-3′ (SEQ ID NO: 6) REG1A-p15′-CGCACGCGTAGGCATCAGCTCTCTACAATTC-3′ (SEQ ID NO: 7) REG1A-p25′-AGCCTCGAGCAGGATCTGAGATAAGAACCACG-3′ (SEQ ID NO: 8) CTRB1-p15′-ACGGCGCTCGAGTCCATCAGTTCTCATC-3′ (SEQ ID NO: 9) CTRB1-p25′-TTACTTAAGCTTGTGTAGGACGCCTGTC-3′ (SEQ ID NO: 10)

These PCR fragments were subcloned into pCRII-TOPO (Invitrogen) togenerate pCRII-TOPO-CCKAR, pCRII-TOPO-uPAR, pCRII-TOPO-RDC1, andpCRII-TOPO-CTRB. All PCR products were verified by sequencing. Thepromoter fragments were then inserted into the KpnI/Xho1 sites of pGL-3basic (Promega, Madison, Wis.) to obtain plasmids pGL3-CCKAR-Luc,pGL3-uPAR-Luc, pGL3-RDC1-Luc, and pGL3-CTRB1-Luc. The CMVenhancer/promoter-controlled Firefly luciferase gene plasmid pCMV-Luc(comprised of the CMV promoter cloned into the pGL-3 vector). Theplasmid pRL-TK, comprising a Renilla luciferase reporter gene, wasobtained from Promega.

The fragment of WPRE enhancer was released from pGEM-3Z-WPRE (a generousgift from Dr. J. B Uney, University of Bristol, Bristol, UK) byAsp718/SalI digestion and inserted into the Small sites of pGL3-basic byblunt ligation to produce intermediate pGL3-Luc-WPRE. Plasmid pGL3-basicwas digested with XbaI, Klenow blunted and annealed to the bluntedAsp718/SalI WPRE fragment of intermediate pGL3-Luc-WPRE to givepGL3-Luc-WPRE. The SpeI/XhoI-blunted CCKAR fragment of pCRII-TOPO-CCKARwas subcloned into blunted NheI/XhoI site of pGL3-Luc-WPRE, resultinginto pGL3-CCKAR-Luc-WPRE. The HindIII/NotI-blunted CCKAR fragment ofpCRII-TOPO-CCKAR was subcloned into the MscI/NheI-blunted site ofpGL3-TSTA-Luc (a gift of Dr. M. Carey, UCLA School of Medicine, LA)(Zhang L, et al, Cancer Res 2003), obtaining pGL3-CCKAR-TSTA-Luc-WPRE.Finally, the CCKAR fragment of pCRII-TOPO-CCKAR digested with NotI/Bg1IIwas inserted into the same site of pGL3-CCKAR-TSTA-Luc-WPRE, producingpGL3-CCKAR-TSTA-Luc-WPRE (pGL3-CTP-Luc).

Transfection

Cells were seeded in 12-well plates at 40-50% confluence at 37° C. with5% CO2 in corresponding medium as described above, 16 h prior totransfection. Cells were transfected with designated plasmid DNA alongwith pRL-TK as internal control, using DOTAP:Chol liposome (from N.Templeton, Baylor College of Medicine, Houston, Tex.) according to therecommended method. The non-expression vector, pGL3-basic, was used as anegative control. To compare the activities of transcriptionalregulatory elements with each other, the same molar amount of plasmidDNA was used.

Orthotopic Animal Models of Pancreatic Cancer and Systemic Plasmid DNADelivery

Athymic female BALB/c nu/nu mice (Charles River Laboratories,Wilmington, Mass.), at 6-8 weeks of age, were used as xenograft hosts.Mice were maintained in a specific pathogen-free environment, incompliance with M.D. Anderson Cancer Center rules. AsPC-1 cells inlogarithmic-phase growth were trypsinized and washed twice with PBS. Forthe orthotopic model, mice were anesthetized with Aventin (Sigma) (Xie,Mol. Endocrinl 2004) and placed at the supine position. The abdomen areawas cleaned with 70% ethanol, and an upper midline abdomen incision wasmade. The pancreas was exteriorized and its tail was injected with 50 μlof aliquots of AsPC-1 cells (1×10⁶ cells). The incision was closed withwound clips.

Plasmid DNA:liposome complexes were prepared as previously described(Templeton, Nat Biotech 1997). Briefly, DNA and DOTOP:Chol stock wereseparately diluted in 5% dextrose in water (D5W) at room temperature.The DNA solution was added rapidly at the surface of the liposomesolution in equal volume and mixed by pipetting up and down twice. Thepreparation was made fresh 2 h prior to injection. The nude mice inwhich tumors reached about 50 mm³ in ectopic model or the same period oftime in othotopic model were injected with 100 μl of DNA:liposomecomplexes containing 50 μg of DNA into the tail vein using a 29-gaugeneedle, once a day for three consecutive days. Mice were in vivo imagedevery day post injection and sacrificed 24 h after last injection.

Luciferase Assays

Transiently transfected cells were lysed and assayed for luciferaseactivity by using the Dual-Luciferase® Reporter Assay System (Promega,Madison, Wis.) following the manufacturer protocol with a TD 20/20luminometer (Turner Designs, Sunnyvale, Calif.). The dual luciferaseratio was defined as the Firefly luciferase activity of the testedplasmids over the Renilla luciferase activity of pRL-TK, expressed asthe means of triplicate transfections, which were repeated at least fourtimes. Compared to the ratio of CMV activity, the percentage waspresented.

To assay tissue-derived luciferase activity, animals were euthanized anddissected. Tissue specimens from tumors and other organs includingpancreas, lung, heart, liver, spleen, kidney, brain, intestine, muscle,and ovary, et al. were resected, and homogenized with a PRO 250homogenizer (Pro Scientific, Inc., Monore, Conn.) in 300 μl ofluciferase lysis buffer (Promega) containing 1/100 diluted proteininhibitor cocktail (Roche). Specimens were centrifuged at 8,000 rpm for5 min and placed temporarily on ice. Luciferase activity of thesupernatants was measured with a Lumat LB9507 luminometer (Berthod, BadWildbad, Germany) and the protein concentration was determined using thedetergent compatible (DC) protein assay system (Bio-Rad, Hercules,Calif.) with MRX microplate reader (Dynex technologies, Inc., Chantilly,Va.). The luminescence results are reported as relative light units(RLU) per milligram of protein.

Imaging and Quantification of Bioluminescence Data

Mice were anaesthetized with Aventin. D-luciferin (Xenogen, Alemeda,Calif.) (30 mg/ml in PBS) was intraperitoneally injected at 150 mg/kgmouse body weight. Ten min after D-luciferin injection, mice were imagedwith an IVISTM Imaging System (Xenogen), consisting of a cooled CCDcamera mounted on a light-tight specimen chamber (dark box), a cameracontroller, a camera cooling system, and a Windows-based computersystem. Imaging parameters were maintained for comparative analysis.Gray scale reflected images and bioluminescence colorized imaged weresuperimposed and analyzed using the Living Imaging software version 2.11(Xenogen). A region of interest (ROI) was manually selected overrelevant regions of signal intensity. The area of the ROI was keptconstant and the intensity was recorded as maximum photon counts withina ROI (Xie et al., 2004). In some experiments after imaging, animalswere euthanized and organs of interest were removed, arranged on black,bioluminescence-free paper, and ex vivo imaged within 30 min.

FIG. 9A illustrates constructs of pancreatic specific promoters. Thepromoter sequences of human Cholecystoskinin A receptor (CCKAR), orphanG protein-coupled receptor (RDC1), urokinase-type plasminogen activatorreceptor (uPAR), and chymotrypsinogen B1 (CTRB1) were PCR-amplified andsubcloned into the reporter plasmid pGL3-basis, thereby regulatingexpression of firefly luciferase gene. Cells were transientlyco-transfected with similar molar quantities of plasmid DNA with theinternal control pRL-TK. As shown in FIG. 9B, forty-eight hours later,dual luciferase ratio was measured and shown as RLU (fold) normalized tothe Rellina luciferase.

Pancreatic Cancer-Specific Expression of a Desired Polynucleotide

FIG. 10A illustrates a schematic diagram of CCKAR-based and CMV-basedconstructs, containing the Firefly luciferase reporter gene under thecontrol of the minimal CCKAR promoter without or with WPRE, orCCKAR/TSTA without or with WPRE, or under CMV enhancer/promoter.Gal4VP2: VP2: duplicated HSV1 VP16 immediate early transactivator domainhave a highly potent to activate when fused to the GAL4 DNA bindingdomain. For the G5E4T sequence (SEQ ID NO:22), G5 comprises 5 tandemcopies of the 17 bp GAL4 DNA binding site near consensus DNA bindingsites. E4T is E4TATA which comprises the adenovirus E4 minimal promoterfrom −38 to +38 relative to the start site. FIG. 10B demonstratesactivity of constructs transiently transfected into AsPC-1, and PANC-1pancreatic cancer cells. Cells were transiently co-transfected withsimilar molar quantities of plasmid DNA with the internal controlpRL-TK. Forty-eight hours later, dual luciferase ratio was measured andthen compared to CMV activity presented as percentage. The datarepresent the mean of four independent experiments; bar, SD. FIG. 10Cshows tissue specificity of CCKAR-based promoter composites. Othercancer cells (LNCaP, PC-3, SKOV3.ip1, MDA-468) and immortalized normalcells (Chang liver, WI-38, 184A1, and E6E7 cells) were transientlyco-transfected with the internal control pRL-TK. Forty-eight hourslater, dual luciferase ratio was measured. The percentage was presentedin comparison to the ratio of the activity in AsPC-1.

In vivo transgene expression in orthotopic tumor model of AsPC cellsafter systemic delivery of CTP-Luc and CMV-Luc plasmid DNA is shown inFIG. 11. FIG. 11A shows in vivo imaging of mice. Nude mice bearingsubcutaneous AsPC-1 tumor were injected in the tail vein with 50 μg ofDNA in DNA (CTP-Luc or CMV-Luc):liposome complexes, once a day for threeconsecutive days. 24 h after last injection, mice were anesthetized, andimaged for 5 min using an IVIS™ Imaging System 10 minutes following i.p.injection of D-luciferin. The representative imaging of mice are shown.FIG. 11B shows firefly luciferase activity in tissue extracts wasquantified with a luminometer and expressed as relative luciferase unitsper milligram of total protein. The ratio was calculated by comparingthe level of luciferase activity of CTP mice to CMV mice.

FIG. 18 shows that human cholecystoskinin type-A receptor (CCKAR)promoter is potentially pancreatic cancer-specific. In FIG. 18A,constructs of candidates for pancreatic cancer-specific promoter. CCKAR,orphan G protein-coupled receptor (RDC1), urokinase-type plasminogenactivator receptor (uPAR), and chymotrypsinogen B1 (CTRB1) werepolymerase chain reaction-amplified and subcloned into the reporterplasmid pGL3-basic, driving a firefly luciferase gene. In FIG. 18B,PANC-1 and AsPC-1 cells were transiently co-transfected with the plasmidDNA indicated and pRL-TK. Forty-eight hours later, the dual luciferaseratio was measured and shown as relative light units (RLU) normalized tothe Renilla luciferase control.

FIG. 19 demonstrates molecular-engineered cholecystoskinin type-Areceptor (CCKAR)-based promoters are more active and retain pancreaticcancer specificity. In FIG. 19A, there is a schematic diagram ofengineered CCKAR-based constructs including pGL3-CCKAR-Luc-WPRE(CCKAR-P-Luc)), pGL3-CCKAR-TSTA-Luc (CCKAR-T-Luc), andpGL3-CCKAR-TSTA-Luc-WPRE (CTP-Luc). In FIG. 19B, there is activity ofCCKAR-based promoters in pancreatic cancer cells. AsPC-1, PANC-1 andPanO2 cells were transiently co-transfected with plasmid DNA and pRL-TK.Forty-eight hours later, the dual luciferase ratio was measured. Thepercentage relative to the activity of the CMV promoter is shown. Thedata represent the mean of four independent experiments. In FIG. 19C,there is tissue specificity of CCKAR-based promoter composites.Non-pancreatic cancer (LNCaP, PC-3, SKOV3.ip1, MDA-MB-468, and HeLa),and normal and immortalized (WI-38, 184A1, and E6E7) cell lines weretransiently co-transfected with the plasmids indicated and pRL-TK.Forty-eight hours later, the dual luciferase ratio was measured. Thepercentage relative to the activity in AsPC-1 cells is shown.

FIG. 20 shows the composite of cholecystokinin-type-A receptor(CCKAR)-two-step-transcriptional activation (TSTA)-woodchuck hepatitisvirus posttranscriptional regulatory element (WPRE), CTP, is robust andpancreatic cancer-specific in an orthotopic animal model. Nude micebearing orthotopic AsPC-1 tumors were give 50 μg of DNA in DNA:liposomecomplexes via the tail vein once a day for 3 consecutive days. In FIG.20A, there is in vivo imaging of mice. Mice were anesthetized and imagedfor 5 min using an IVIS imaging system 10 minutes after intraperitonealinjection of D-luciferin. In FIG. 20B, there is tissue distribution ofluciferase expression. Tissue specimens from tumors and organs as shownwere dissected and measured for luciferase activity with a luminometer.Data were expressed as relative luciferase units per milligram of totalprotein.

FIG. 21 provides expression of Bik mutant (BikDD) driven by CTP killspancreatic cancer cells effectively and specifically. In FIG. 20A, thereis a schematic diagram of expression constructs in the pUK21 backbone.CMV-BikDD, pUK21-CMV-BikDD; CTP-BikDD, pUK21-CTP-BikDD. In FIG. 20B, thekilling effects of BikDD driven by CMV or CTP are provided. A panel ofpancreatic cancer (AsPC-1, PANC-1, MDA-Panc28, and PanO2), immortalizedhuman pancreatic epithelial E6E7 cells were co-transfected with 2 μg ofpUK21 (negative control), pUK21-CMV-BikDD (positive control), orpUK21-CTP-BikDD, plus 100 ng of pGL3-CMV-Luc. Forty-eight hours aftertransfection, the luciferase activity was imaged for 2 min using an IVISimaging system following a 5-minute incubation with 5 ng/ml ofD-luciferin. Representative images were shown in the upper panel. Thepercentage of the signal compared with the negative control (set as100%) was calculated (lower panel).

In particular embodiments of the present invention, constructs aresimilarly generated comprising these exemplary pancreatic-specificpromoters operatively linked to a polynucleotide encoding a mutant Bik,followed by introduction into a mammal in need of pancreatic cancertherapy treatment based on analogous methods described herein.Parameters are easily optimized by those of skill in the art, such asdelivery mode, concentration of composition, and so forth.

In further embodiments of the present invention, the pancreaticcancer-specific elements are narrowed further to identify even smallersegments within that retain pancreatic cancer-specific expressionactivity. For example, deletion constructs may be made of theserespective regions, and their tissue specificity is tested to identifythe smaller segments that maintain the ability to direct expression inpancreatic cancer tissue.

Example 3 Prostate Cancer-Specific Expression

The present inventors utilized prostate cancer-specific promotersequences to control expression of a polynucleotide encoding a mutantBik polypeptide. Exemplary methods and compositions directed to thisgoal are described in this Example.

Development of Targeted Gene Therapy for Metastatic and RecurrentHormonal Refractory Prostate Cancer

In specific embodiments, a promoter that regulates expression of mutantBik in both androgen-dependent and androgen-independent manners isutilized. A skilled artisan recognizes that in prostate cancer genetherapy, prostate specific promoters, like PSA (Greenberg, DeMayo etal., 1995; Spitzweg, Zhang et al., 1999; Latham, Searle et al., 2000;Wu, Matherly et al., 2001), probasin (Greenberg, DeMayo et al., 1995;Zhang, Thomas et al., 2000; Wen, Giri et al., 2003) and hK2 (Xie, Zhaoet al., 2001) have been recently developed. The activities of thesepromoters are androgen-dependent. For patients with advanced, metastaticprostate cancer, hormonal ablation is the primary choice of therapy.However, after chemical castration, the adrenal glands secrete largeamounts of the inactive precursor steroids dehydroepiandrosterone andandrostenedione as a compensatory source of androgen (Denis, 1996).Furthermore, most androgen-independent prostate adenocarcinoma cellsup-regulate AR expression, select for mutant AR with increased steroidresponsiveness or reduced steroid specificity, or up-regulate growthfactor signaling that can stimulate AR activity (Feldman and Feldman,2001). Moreover, significant side effects associated with this therapyhave led to the more widespread use of intermittent ablation protocols,in which patients are treated with leutinizing hormone releasing hormoneagonists for up to 12 months before discontinuing therapy until tumorprogression begins to occur (Bruchovsky, Klotz et al. 2000). Thus, fornumerous disease stages, patients are often hormonally intact, allowingthe use of androgen-responsive vectors to direct expression oftherapeutic genes to prostatic tissue.

Androgen-independent prostate cancer (AIPC) is an untreatable form ofprostate cancer in which the normal dependence on androgens for growthand survival has been bypassed. AIPC is selected for by androgenablation therapy (Feldman and Feldman, 2001). It is proposed thatmalignant androgen- and AR-independent epithelial stem cells ‘lurking’in the normal prostate become selected for by therapy. A subset of theseAR mutations map to the ligand-binding domain (LBD) and are proposed tocause resistance by altering the response of the receptor such thatnoncanonical ligands such as estrogen or hydrocortisone, or evenandrogen receptor antagonists such as flutamide, behave as agonists(Chen, Welsbie et al. 2004). Under these conditions, it is very criticalfor developing an androgen-independent promoter in suicide gene therapyto completely eradicate metastatic and recurrent hormonal refractoryprostate cancer.

The Human Telomerase Reverse Transcriptase (hTERT) Promoter isCancer-Specific

Over the past several years, the enzyme telomerase reversetranscriptase, responsible for maintaining the telomeric DNA at the endof chromosomes, has been the subject of experimental findings thatassociate it with a magic bullet against cancer. Telomeres are essentialelement that protects chromosome ends from degradation and end-to-endfusions, rearrangements and chromosome loss. The absence of telomeraseactivity in human somatic cells prevents compensation for the loss oftelomeric DNA ensuing from the inability of conventional DNA polymeraseto fully replicate linear DNA molecule. The resulting shortening of thetelomeres limits the cell proliferative lifespan and cell senescence.Cell immortalization in vitro and tumor progression are associated with,and may depend on, activation of telomerase.

There has been much in the literature regarding telomerase activity indifferent patients with cancer. For example, generally telomeraseexpression was found in 80-100% of human cancer, but almost all normalcells are negative except for sperm cells or highly proliferating cells.In prostate cancer, about 84% of prostate carcinomas and 100% humanprostate cancer cell lines are hTERT-positive (Vasef, Ross et al. 1999).Recently, the hTERT promoter has been used for cancer gene therapy, aswell as prostate cancer gene therapy (Gu, Andreeff et al. 2002; Kim, Kimet al. 2003; Irving, Wang et al. 2004).

Two-Step Transcription Amplification (TSTA) Significantly Increases GeneExpression

The hTERT promoter increases the safety and effectiveness of genetherapy. However, the activity of this unmodified hTERT promoter is muchweaker than that of commonly used non-tissue-specific virus-basedpromoters, such as the cytomegalovirus (CMV) promoter (Cong, Wen et al.1999; Gu, Andreeff et al., 2002; Komata, Kondo et al. 2002). One of theamplification approaches using the GAL4-VP16 fusion protein, called atwo-step transcriptional amplification (activation) (TSTA) approach, canpotentially be used to augment the transcriptional activity of cellularpromoters (Iyer, Wu et al. 2001; Zhang, Adams et al. 2002). In thissystem, the first step involves the tissue-specific expression of theGAL4-VP16 fusion protein. In the second step, GAL4-VP16, in turn, drivestarget gene expression under the control of GAL4 response elements in aminimal promoter. The use of TSTA can potentially lead to amplifiedlevels of the transgene expression.

WPRE is a Useful Enhancer

To increase the activity of tissue-specific promoters, the presentinventors and others used CMV enhancer fused to the minimaltissue-specific promoter. Though the activity was increased, the tissuespecificity was decreased (unpublished and (Latham, Searle et al.,2000)). To address this issue, the present inventors utilized thepost-transcriptional regulatory element of the woodchuck hepatitis virus(WPRE), which involves modification of RNA polyadenylation, RNA export,and/or RNA translation (Donello, Loeb et al., 1998). Enhancement of WPREoccurred both during transient expression in non-viral vectors and viralvectors (Loeb, Cordier et al., 1999; Glover, Bienemann et al. 2002) andwhen the gene is stably incorporated into the genome of target cellswith no loss of tissue specificity (Lipshutz, Titre et al. 2003). WPREin the sense orientation cloned between the target gene and the poly(A)sequence stimulated 2- to 7-fold more luciferase expression in vitro and2- to 50-fold in vivo without the use of the WPRE (Zufferey, Donello etal., 1999; Lipshutz, Titre et al., 2003). Furthermore, long-termtransgene expression can be mediated by WPRE-containing adenoviralvectors (Glover, Bienemann et al., 2003). Therefore, the WPRE is aneffective tool for increasing and prolonging the expression oftransgenes in gene therapy.

TSTA and WPRE Enhance the Activity of hTERT Promoter

To determine whether TSTA and WPRE enhance the activity of hTERTpromoter, the present inventors first subcloned a series of TSTA- andWPRE-containing hTERTp-based promoter composites. The hTERTp fragment(nt −378 to +56) (Takakura, Kyo et al., 1999) was PCR-amplified from theDNA extracts of LNCaP cells. The hTERTp fragment was subcloned intopGL3-Basic plasmid to drive the firefly luciferase gene, leading tophTERTp-Luc. The WPRE was then inserted into phTERTp-Luc-Luc, resultingin phTERTp-Luc-WPRE. To employ the TSTA system, hTERTp was substitutedfor PSA promoter of pTSTA plasmid (Zhang, Johnson et al., 2003),producing phTERTp-TSTA-Luc. Finally, the plasmid phTERTp-TSTA-Luc-WPREwas obtained by inserting the WPRE fragment into phTERTp-TSTA-Luc.

ARR2-Fused hTERTp-Based Promoters can be Stimulated by Androgen

The activity of the different promoter composites are tested inAR-positive and AR-negative prostate cancer cells, as well as normalhuman endothelial cells (HUVEC), which were transiently co-transfectedwith similar molar quantities of plasmid DNA with the internal controlpRL-TK. pGL3-Basic without enhancer/promoter was used for negativecontrol. Forty-eight hours later, dual luciferase ratio was measured andthen compared to CMV activity presented as percentage.

In particular, LNCaP cells or PC-3 cells were transiently co-transfectedwith similar molar quantities of plasmid DNA with the internal controlpRL-TK. Forty-eight hours later; dual luciferase ratio was measured andthen compared to CMV activity presented as percentage. The datarepresent means of four independent experiments; bar, SD. FIG. 12C showstissue specificity of hTERTp-based composites. The lung fibroblast cellsWI-38 were transiently co-transfected with the indicated plasmids andthe internal control pRL-TK. Forty-eight hours later, dual luciferaseratio was measured. The percentage was presented in comparison to theratio of the activity in AsPC-1.

As shown in FIG. 12, the hTERTp is active in both LNCaP cell and PC-3cells, but its activity was very weak compared to CMV enhancer/promoter.However, WPRE increased the activity by about 2-fold. Surprisingly, TSTAsystem can boost the activity to 67% of CMV activity in LNCaP cell and90% in PC-3 cells. For the TSTA system in combination with WPRE, theactivity is comparable to CMV in PC-3 and is even 1.5-fold higher inLNCaP cells. In contrast, its activity remains undetectable in WI-38cells (data not shown).

In most cases of recurrent prostate cancers, the AR gene is amplifiedand/or AR is overexpressed (Visakorpi T, Nat Genet 1995 (Chen, Welsbieet al. 2004)). Therefore, it should also greatly improve the effectiveindex if the activity of this system can be stimulated by androgen. Toaccomplish this goal, the ARR2 element derived from plasmid ARR2PB(Zhang, Thomas et al. 2000; Xie, Zhao et a/2001) was fused to the hTERTppromoter of phTERTp-TSTA-Luc and phTERTp-TSTA-Luc-WPRE, to produceplasmid pARR2.hTERTp-TSTA-Luc and pARR2.hTERTp-TSTA-Luc-WPRE (FIG. 13A).LNCaP cells and PC-3 cells were transiently co-transfected with similarmolar quantities of plasmid DNA with the internal control pRL-TK andstimulated with 0.01, 0.1, 1, and 10 nM concentrations of nonmetabolizedandrogen analog R1881 for two days in medium containingcharcoal/dextran-treated FBS. Thereafter, dual luciferase ratio wasmeasured and then compared to CMV activity presented as percentage, asdescribed above. As expected, the activity of ARR2.hTERTp-TSTA andARR2.hTERTp-TSTA-WPRE composites was increased in an androgen-dependentmanner by 10-fold higher than CMV in LNCaP cells, without there beingsignificant change in PC-3 cells.

In further embodiments of the present invention, the respective prostatecancer-specific elements are narrowed further to identify even smallersegments within that retain prostate cancer-specific expressionactivity. For example, deletion constructs may be made of theserespective regions, and their tissue specificity is tested to identifythe smaller segments that maintain the ability to direct expression inprostate cancer tissue.

TSTA and WPRE Enhance the Activity of the hTERT Promoter

In FIG. 22, there is comparison of firefly luciferase activity under thecontrol of CMV promoter and hTERTp-based promoter composites. FIG. 22Ashows a schematic diagram of reporter constructs. FIG. 22B shows thatprostate cancer LNCaP and PC-3, and normal human fibroblast WI-38 cellslines were transiently co-transfected with reporter plasmid DNA and theinternal control vector pRL-TK. 48 h later, the dual luciferase ratiowas measured. Shown are the luciferase activities (folds) in relative tothe CMV promoter (setting at 1). The hTERTp activity is increased inboth LNCaP and PC-3 cells through the TSTA system, and is furtherenhanced by WPRE (FIG. 22B). In combination with TSTA and WPRE, theactivity of hTERTp-TSTA-WPRE is comparable to or even 1.5-fold greaterthan that of the CMV promoter, in PC-3 and LNCaP cells, respectively.Importantly, its activity remains silent in human normal lung fibroblastcells WI-38 and in normal tissue of the mouse model further confirmedlater.

The cis-acting ARR2 element further boosts the activity of TSTA- andWPRE-modified hTERTp in response to androgen stimulation in vivo. Inmost cases of recurrent or metastatic prostate cancer through ADPC toAIPC, the AR gene is amplified and/or AR is overexpressed and still ableto bind to androgen (or androgen analog) following by binding to theandrogen responsive element (ARE), resulting in transcriptionalactivation (Chen et al., 2004; Visakorpi et al., 1995). In this regard,the therapeutic index should be greatly improved if the promotercontains an ARE, which binds to the androgen (or androgen analog)/ARcomplexes, leading to stimulation of the therapeutic gene expression.

To accomplish this goal, the ARR2 element (androgen-receptor responsiveelement 2), which is derived from ARR2PB (Xie et al., 2001; Zhang etal., 2000), was fused to upstream of hTERTp in our newly constructedsystems, to generate ATT-Luc (pGl3-ARR2.hTERTp-TSTA-Luc) and ATTP-Luc(pGl3-ARR2.hTERTp-TSTA-Luc-WPRE) (FIG. 23A). ARR2PB-Luc(pGl3-ARR2PB-Luc) was used as a control and CMV-Luc as a reference tool.The cells were transiently co-transfected with the constructs and theinternal control vector pRL-TK and incubated with increasingconcentrations of androgen analog, R1881. Indeed, the activities of ATTand ATTP composites were increased in an androgen-dependent manner, upto 15- and 25-fold greater in AR+ADPC LNCaP cells and up to 2.8- and5.5-fold in AR+AIPC LAPC-4 cells, respectively, than that of the CMVpromoter (FIG. 23B). ARR2 does not interfere with the transcriptionalactivities of hTERTp-TSTA and hTERTp-TSTA-WPRE in PC-3 and LNCaP cells(FIG. 22B and FIG. 23B), and their specificity in normal cells (data notshown). Compared with ATTP and ATT, ARR2PB is much less active in AR+(LNCaP and LAPC-4) and almost inactive in AR− (PC-3 and DU145) cells(FIG. 23B). Thus, the newly generated ATTP and ATT are highly active inall four cell lines tested, which activities are comparable in AR− AIPC(PC-3 and DU145) cells to, and much stronger in AR+ ADPC (LNCaP) and AR+AIPC(LAPC-4) cells than that of the CMV promoter, and importantly remainsilent in the normal cells.

ATTP is robust in ADPC and AIPC xenografts in vivo. To further determinewhether the activity and specificity of ATTP would be maintained invivo, the present inventors established male BABL/c nu/nu mouse modelsof s.c. LNCaP and PC-3 xenografts. Mice bearing LNCaP or PC-3 tumorswere i.v. injected with 50 μg of pGL3-ATTP-Luc and pGL3-CMV-Luc inDNA:liposome complexes, once a day for three consecutive days.Mice-bearing LNCaP tumors were in vivo imaged with a non-invasiveimaging system (Xie et al., 2004) for two minutes every day andsacrificed 24 h after the last injection. Bioluminescent imaging showedvery brilliant light spots in the area of the thorax (lung/heart) ofmice treated with CMV-Luc, but almost none in the same area of micetreated with ATTP-Luc (FIG. 24A). To further characterize the source ofthe light, the mice were sacrificed immediately after live imaging, andtheir major organs were dissected to be imaged ex vivo. The presentinventors verified that the strongest photo-emitting organ was the lungof mice treated with CMV-Luc, whereas the signal from the lungs of micetreated with ATTP-Luc was undetectable (FIG. 24B). To increase signalstrength by prolonging photon-exciting time, the dissected tumors werethen immediately imaged for 10 min. The tumors from mice treated withATTP-Luc produced much stronger signal than ones from mice treated withCMV-Luc (P, 0.002) (FIG. 24C). Consistent with the in vivo and ex vivoimaging results, the luciferase activities from lungs and hearts of micetreated with CMV-Luc were significantly greater than that of micetreated with ATTP-Luc. In contrast, the luciferase activity from tumorsof mice treated with ATTP-Luc was 14.5-fold greater than that of thetumors of mice with CMV-Luc (P, 0.004) (FIG. 24E). The cancer-specificindex (the luciferase activity of tumors to lung) (Chen et al., 2004)was 14.3 for ATTP-Luc in contrast to 0.012 for CMV-Luc in the LNCaPtumor model. Due to the fact that PC-3 cells are AR− the signal fromPC-3 tumors treated with ATTP-Luc was not as great as that from LNCaPtumors. However, the signal from the PC-3 tumors of mice treated withATTP-Luc was still stronger than that of mice treated with CMV-Luc (FIG.24D) and the cancer-specific index (3.9) of ATTP-Luc is still muchbetter that that (0.015) of CMV-Luc (data not shown). Taken together,the “chimeric” ATTP is able to direct a gene of interest to the prostatetumor (both AR+ and AR−) at least as efficiently as that of the CMVpromoter in the AR− prostate cancer, and much more efficiently in AR+prostate cancer whereas there is almost no expression in normal cells.

Example 4 In Vitro Testing of Cancer-Specific Promoters

A construct(s) comprising the inventive promoters operably linked to arespective therapeutic polynucleotide are tested in vitro. For example,the control sequences are selected, in some embodiments based onpreviously generated data suggesting the sequence is effective in adesired tissue or cell. In other embodiments, control sequences areselected without prior knowledge of potential effectiveness. The controlsequence to be tested is operably linked to a reporter sequence, such asone whose expression and/or gene product may be monitored, including bycolor, light, or fluorescence, for example. Examples of reporter genesinclude luciferase or β-galactosidase. Additional control sequences ofany kind may also be added to the construct, including transcriptionalor post-transcriptional control sequences, minimal promoters, and soforth. The construct to be tested and its one or more appropriatecontrols are then introduced into a desired cell and assayed forexpression. In particular embodiments, the construct to be testedgenerates expression at such levels as determined by the skilled artisanto be effective in the desired cell or tissue in which it resides.

Example 5 In Vivo Testing of Cancer-Specific Promoters

A construct(s) comprising the inventive promoters operably linked to arespective therapeutic polynucleotide as it relates to its anti-tumoractivity is tested in an animal study. The construct is delivered by avector, such as in a liposome or on a plasmid or viral vector, into nudemice models to test for its anti-tumor activity. Once the anti-tumoractivity is demonstrated, potential toxicity is further examined usingimmunocompetent mice, followed by clinical trials.

In a specific embodiment, the preferential growth inhibitory activity ofa construct comprising the inventive promoter operably linked to atherapeutic polynucleotide is tested in an animal. Briefly, and byexample only, HER-2/neu overexpressing breast cancer cell lines (such asSKBR3 and MDA-MB361) are administered into mammary fat-pad of nude miceto generate a breast xenografted model. After the tumors reach aparticular size, the construct of the present invention or its controlis intravenously injected into the mouse in an admixture with anacceptable carrier, such as liposomes. The tumor sizes and survivalcurve from these treatments are compared and statistically analyzed. Ina preferred embodiment, the constructs comprising the promoters of theinvention preferentially inhibit the growth of a tumortissue-specifically compared to that of wild-type p21.

Example 6 Clinical Testing with Cancer-Specific Promoters

This example is concerned with the development of human treatmentprotocols using constructs comprising the cancer-specific promoters ofthe invention alone or in combination with other anti-cancer drugs. Theanti-cancer drug treatment using constructs comprising thecancer-specific promoters of the invention will be of use in theclinical treatment of various cancers. Such treatment will beparticularly useful tools in anti-tumor therapy, for example, intreating patients with the respective breast, prostate, and pancreaticcancers, such as those that are resistant to conventionalchemotherapeutic regimens.

The various elements of conducting a clinical trial, including patienttreatment and monitoring, will be known to those of skill in the art inlight of the present disclosure. The following information is beingpresented as a general guideline for use in establishing the constructscomprising the cancer-specific promoters of the invention in clinicaltrials.

Patients with advanced, metastatic breast, prostate, or pancreaticcancers chosen for clinical study will typically be at high risk fordeveloping the cancer, will have been treated previously for the cancerwhich is presently in remission, or will have failed to respond to atleast one course of conventional therapy. In an exemplary clinicalprotocol, patients may undergo placement of a Tenckhoff catheter, orother suitable device, in the pleural or peritoneal cavity and undergoserial sampling of pleural/peritoneal effusion. Typically, one will wishto determine the absence of known loculation of the pleural orperitoneal cavity, creatinine levels that are below 2 mg/dl, andbilirubin levels that are below 2 mg/dl. The patient should exhibit anormal coagulation profile.

In regard to the constructs comprising the cancer-specific promoters ofthe invention and other anti-cancer drug administration, a Tenckhoffcatheter, or alternative device may be placed in the pleural cavity orin the peritoneal cavity, unless such a device is already in place fromprior surgery. A sample of pleural or peritoneal fluid can be obtained,so that baseline cellularity, cytology, LDH, and appropriate markers inthe fluid (CEA, CA15-3, CA 125, PSA, p38 (phosphorylated andun-phosphorylated forms), Akt (phosphorylated and un-phosphorylatedforms) and in the cells (constructs comprising the cancer-specificpromoters of the invention) may be assessed and recorded.

In the same procedure, the constructs comprising the cancer-specificpromoters of the invention may be administered alone or in combinationwith the other anti-cancer drug. The administration may be in thepleural/peritoneal cavity, directly into the tumor, or in a systemicmanner, for example. The starting dose may be about 0.05 mg/kg bodyweight. Three patients may be treated at each dose level in the absenceof grade>3 toxicity. Dose escalation may be done by 100% increments (0.5mg, 1 mg, 2 mg, 4 mg) until drug related grade 2 toxicity is detected.Thereafter dose escalation may proceed by 25% increments. Theadministered dose may be fractionated equally into two infusions,separated by six hours if the combined endotoxin levels determined forthe lot of the constructs comprising the cancer-specific promoters ofthe invention, and the lot of anti-cancer drug exceed 5 EU/kg for anygiven patient.

The constructs comprising the cancer-specific promoters of the inventionand/or the other anti-cancer drug combination, may be administered overa short infusion time or at a steady rate of infusion over a 7 to 21 dayperiod. The constructs comprising the cancer-specific promoters of theinvention infusion may be administered alone or in combination with theanti-cancer drug. The infusion given at any dose level will be dependentupon the toxicity achieved after each. Hence, if Grade II toxicity wasreached after any single infusion, or at a particular period of time fora steady rate infusion, further doses should be withheld or the steadyrate infusion stopped unless toxicity improved. Increasing doses of theconstructs comprising the cancer-specific promoters of the invention, incombination with an anti-cancer drug will be administered to groups ofpatients until approximately 60% of patients show unacceptable Grade IIIor IV toxicity in any category. Doses that are ⅔ of this value could bedefined as the safe dose.

Physical examination, tumor measurements, and laboratory tests should,of course, be performed before treatment and at intervals of about 3-4weeks later. Laboratory studies should include CBC, differential andplatelet count, urinalysis, SMA-12-100 (liver and renal function tests),coagulation profile, and any other appropriate chemistry studies todetermine the extent of disease, or determine the cause of existingsymptoms. Also appropriate biological markers in serum should bemonitored e.g. CEA, CA 15-3, p38 (phosphorylated and non-phosphorylatedforms) and Akt (phosphorylated and non-phosphorylated forms), p185, etc.

To monitor disease course and evaluate the anti-tumor responses, it iscontemplated that the patients should be examined for appropriate tumormarkers every 4 weeks, if initially abnormal, with twice weekly CBC,differential and platelet count for the 4 weeks; then, if nomyelosuppression has been observed, weekly. If any patient has prolongedmyelosuppression, a bone marrow examination is advised to rule out thepossibility of tumor invasion of the marrow as the cause ofpancytopenia. Coagulation profile shall be obtained every 4 weeks. AnSMA-12-100 shall be performed weekly. Pleural/peritoneal effusion may besampled 72 hours after the first dose, weekly thereafter for the firsttwo courses, then every 4 weeks until progression or off study.Cellularity, cytology, LDH, and appropriate markers in the fluid (CEA,CA15-3, CA 125, ki67 and Tunel assay to measure apoptosis, Akt) and inthe cells (Akt) may be assessed. When measurable disease is present,tumor measurements are to be recorded every 4 weeks. Appropriateradiological studies should be repeated every 8 weeks to evaluate tumorresponse. Spirometry and DLCO may be repeated 4 and 8 weeks afterinitiation of therapy and at the time study participation ends. Anurinalysis may be performed every 4 weeks.

Clinical responses may be defined by acceptable measure. For example, acomplete response may be defined by the disappearance of all measurabledisease for at least a month. Whereas a partial response may be definedby a 50% or greater reduction of the sum of the products ofperpendicular diameters of all evaluable tumor nodules or at least 1month with no tumor sites showing enlargement. Similarly, a mixedresponse may be defined by a reduction of the product of perpendiculardiameters of all measurable lesions by 50% or greater with progressionin one or more sites.

REFERENCES

All patents and publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference in their entirety to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

PATENTS

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1.-16. (canceled)
 17. A polynucleotide construct comprising a prostatecancer-specific control sequence, said control sequence comprising atleast two of the following sequences: a prostate tissue-specific controlsequence; a cancer-specific control sequence; and a two-steptranscriptional amplification (TSTA) sequence, said TSTA sequenceincluding a DNA binding domain and an activation domain.
 18. Theconstruct of claim 17, wherein said prostate tissue-specific controlsequence comprises SEQ ID NO:17.
 19. The construct of claim 17, whereinsaid cancer-specific control sequence comprises SEQ ID NO:18.
 20. Theconstruct of claim 17, wherein the DNA binding domain is Gal1, Gal4, orLexA.
 21. The construct of claim 17, wherein the activation domain isVP2 or VP16.
 22. The construct of claim 17, wherein the TSTA sequence isGAL4-VP2 or GAL4-VP16.
 23. The construct of claim 17, further comprisinga post-transcriptional regulatory sequence.
 24. The construct of claim23, wherein the post-transcriptional regulatory sequence is a woodchuckhepatitis virus post-transcriptional regulatory element (WPRE) sequence.25. The construct of claim 17, wherein said control sequence is operablylinked to a polynucleotide encoding a therapeutic gene product.
 26. Theconstruct of claim 25, wherein the therapeutic gene product is aninhibitor of cell proliferation, a regulator of programmed cell death,or a tumor suppressor.
 27. The construct of claim 17, further defined asbeing comprised in a liposome. 28.-46. (canceled)